Methods of degrading or hydrolyzing a polysaccharide

ABSTRACT

The invention provides a method of degrading or hydrolyzing a polysaccharide, preferably cellulose or chitin, comprising contacting said polysaccharide with one or more oxidohydrolytic enzymes, preferably a CBM33 family protein (preferably CBP21) or a GH61 family protein, wherein said degradation or hydrolysis is carried out in the presence of at least one reducing agent and at least one divalent metal ion. A method of producing an organic substance comprising said method is also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 15/660,027 filed on Jul. 26, 2017, now U.S. Pat. No. 10,273,512,which is a divisional of U.S. application Ser. No. 13/814,450 filed onFeb. 5, 2013, now U.S. Pat. No. 9,758,802, which is a 35 U.S.C. § 371national application of PCT/US2011/046838 filed on Nov. 5, 2011, whichclaims priority or the benefit under 35 U.S.C. § 119 of GB ApplicationNo. 1105062.2 filed on Mar. 25, 2011, GB Application No. 1016858.1 filedon Oct. 6, 2010, and GB Application No. 1013317.1 filed on Aug. 6, 2010,the contents of which are fully incorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to methods of degrading or hydrolyzing apolysaccharide, such as chitin or cellulose, comprising contacting saidpolysaccharide with an oxidohydrolytic enzyme, such as CBP21 or a GH61protein, wherein said degradation or hydrolysis is carried out in thepresence of at least one reducing agent and at least one divalent metalion. The invention also extends to the use of additional saccharolyticenzymes such as hydrolases and beta-glucosidases to increase the levelor extent of degradation and to fermentation of the resulting sugars togenerate an organic substance such as an alcohol, preferably ethanol,which may be used as a biofuel.

Description of the Related Art

Efficient enzymatic conversion of crystalline polysaccharides is crucialfor an economically and environmentally sustainable bioeconomy, butremains unfavourably inefficient.

The transition to a more environmental friendly economy has spurredresearch on enzymes capable of efficiently degrading recalcitrantcarbohydrates, such as cellulose and chitin (FIG. 1A), for theproduction of biofuels (Himmel et al., 2007, Science 315: 804).Cellulose is the most abundant organic molecule on the earth and offersa renewable and seemingly inexhaustible feedstock for the production offuels and chemicals. Chitin is a common constituent of fungal cellwalls, shells of crustaceans and exoskeletons of insects. It is thesecond most abundant polymer in nature and each year more than onebillion tons of chitin are produced in the biosphere, mainly by insects,fungi, crustaceans and other marine organisms. Chitin is abundantlyavailable as a by-product from aquaculture, one of the fastest growingbioproduction industries on earth.

The conversion of cellulosic feedstocks into ethanol has the advantagesof the ready availability of large amounts of feedstock, thedesirability of avoiding burning or land filling the materials and thecleanness of the ethanol fuel. Wood, agricultural residues, herbaceouscrops and municipal solid wastes have been considered as feedstocks forethanol production. These materials primarily consist of cellulose,hemicellulose and the non-polysaccharide lignin. Once the cellulose isconverted to glucose, the glucose is easily fermented by yeast intoethanol.

A variety of microorganisms exist for fermenting the products ofhydrolysis of polysaccharides to yield desirable end products such asalcohol. Selection of appropriate microorganisms allows the products ofhydrolysis of cellulose, chitin and other polysaccharides to befermented to yield useful products, such as alcohol.

The predominant polysaccharide in the primary cell wall of biomass iscellulose, the second most abundant is hemi-cellulose and the third ispectin. The secondary cell wall, produced after the cell has stoppedgrowing, also contains polysaccharides and is strengthened by polymericlignin covalently cross-linked to hemicellulose. Cellulose is ahomopolymer of anhydrocellobiose and thus a linear beta-(1-4)-D-glucan,while hemicelluloses include a variety of compounds, such as xylans,xyloglucans, arabinoxylans and mannans in complex branched structureswith a spectrum of substituents. Although generally polymorphous,cellulose is found in plant tissue primarily as an insoluble crystallinematrix of parallel glucan chains. Hemicelluloses usually hydrogen bondto cellulose, as well as to other hemicelluloses, which help stabilizethe cell wall matrix.

Bacteria and fungi have evolved complex enzymatic systems enabling theirgrowth on plant material rich in cellulose, but these organismstypically require weeks, months or even years to decompose a fallen logor a tilled corn stalk. Likewise, microorganisms contain enzymaticsystems for degrading chitin. Bacterial chitinase helps to provide acarbon source for bacterial growth. Insects produce chitinase to digesttheir cuticle at each molt. In plants, chitinase is thought to provide aprotective role against parasitic fungi. For chemical or fuel productionfrom these same cellulose- and chitin-containing materials, industryrequires affordable chemical or enzymatic systems that can do the job inhours or in days.

Traditionally, enzyme systems capable of degrading such carbohydrateswere considered to consist of two types of hydrolytic enzymes calledglycoside hydrolases: endo-acting enzymes that cut randomly in thecarbohydrate chain and processive exo-acting enzymes (chito- orcellobiohydrolases), which degrade the polymers from chain ends (FIG.1B). Although this model is generally accepted, it remains difficult tounderstand how, e.g., an endoglucanase would be capable of pulling asingle polysaccharide chain out of its crystalline environment andforcing the chain productively into its active site cleft (FIG. 1B).

Ever since cellulases caught the interest of biochemists, there havebeen speculations about the possible existence of a substrate-disruptingfactor that could make the crystalline substrate more accessible tohydrolytic enzyme (Reese et al., 1950, J Bacteriol. 59: 485). Recently,it was discovered that microorganisms that breakdown chitin indeedproduce a protein that increases substrate accessibility and potentiateshydrolytic enzymes (Vaaje-Kolstad et al., 2005, J. Biol. Chem. 280:28492; FIGS. 1C and 1D). The first example was a single-domain proteincalled CBP21 (CBP for Chitin-Binding Protein) produced by thechitinolytic bacterium Serratia marcescens. This protein has beenclassified as carbohydrate-binding module (CBM) and belongs to familyCBM33 as defined by the CAZy nomenclature (Boraston et al., 2004,Biochem. J. 382: 769, Henrissat, 1991, Biochem. J. 280 (Pt 2): 309).Another example concerns two CBM33-containing proteins from Thermobifidafusca, called E7 and E8, which potentiate chitin hydrolysis by chitinaseand cellulose hydrolysis by cellulases (Moser et al., 2008, Biotechnol.Bioeng. 100(6): 1066-77). Like CBP21, E7 is a single domain protein onlycomprising a CBM33 domain. E8 is a three-domain protein, meaning that itcarries two domains in addition to a CBM33 domain.

It has recently been shown that proteins presently classified as family61 glycoside hydrolases (GH61) in the CAZy nomenclature actsynergistically with cellulases (Harris et al., 2010, Biochemistry 49:3305) and are structurally similar to CBM33 proteins (Harris et al.,2010, supra; Karkehabadi et al., 2008, J. Mol. Biol. 383: 144; FIG. 1E).While CBM33 and GH61 have little sequence similarity, the structuralsimilarity is evident (FIGS. 1D and 1E), including a diagnostic fullyconserved arrangement of the N-terminal amino group, an N-terminalhistidine and one other histidine residue (FIG. 1F; Karkehabadi et al.,2008, supra) forming a promiscuous metal-binding site (see below). Onthe basis of available literature data, including a recent comprehensivestudy of several GH61 proteins, it seems highly unlikely that GH61proteins are endoglucanases, as originally thought (Harris et al., 2010,supra). Like CBM33 proteins, GH61 proteins do not have asubstrate-binding cleft or pocket, nor do they possess a characteristicarrangement of acidic amino acids that could indicate a glycosidehydrolase activity. Instead, both types of proteins show an almost flatsubstrate-binding surface (FIGS. 1D and 1E; Harris et al., 2010, supra;Vaaje-Kolstad et al., 2005, J. Biol. Chem. 280: 11313). All in all,these results and observations show that CBM33 and GH61 proteins havesimilar functions, i.e., potentiating the efficacy of known hydrolyticenzymes (glycoside hydrolases) acting on crystalline polysaccharides.Furthermore, these results and observations strongly suggest that theseproteins do so employing the same type of mechanism. So far, thismechanism has remained elusive.

The present invention provides methods of degrading or hydrolyzing apolysaccharide, such as chitin or cellulose, comprising contacting saidpolysaccharide with an oxidohydrolytic enzyme in the presence of atleast one reducing agent and at least one divalent metal ion.

SUMMARY OF THE INVENTION

The present invention relates to methods of degrading or hydrolyzing apolysaccharide comprising contacting said polysaccharide with one ormore oxidohydrolytic enzymes, wherein said degradation or hydrolysis iscarried out in the presence of at least one reducing agent and at leastone divalent metal ion.

The present invention also relates to methods of producing solublesaccharides, wherein said method comprises degrading or hydrolyzing apolysaccharide by the method defined above, wherein said degradation orhydrolysis releases said soluble saccharides and optionally isolatingsaid soluble saccharides.

The present invention also relates to methods of producing an organicsubstance, comprising the steps of: (i) degrading or hydrolyzing apolysaccharide by the method defined above to produce a solutioncomprising soluble saccharides; (ii) fermenting said solublesaccharides, to produce said organic substance as the fermentationproduct; and optionally, (iii) recovering said organic substance.

The present invention also relates to a process for producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial with an enzyme composition comprising an endoglucanase, acellobiohydrolase, a beta-glucosidase, a GH61 polypeptide havingcellulolytic enhancing activity, and a CBM33; (b) fermenting thesaccharified cellulosic material with one or more fermentingmicroorganisms to produce the fermentation product; and (c) recoveringthe fermentation product from the fermentation.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows repetitive disaccharide units in cellulose and chitin.FIG. 1B shows a schematic representation of the degradation of chitin orcellulose by endo-acting (top) and (processive) exo-acting glycosidehydrolases (bottom). FIG. 1C shows the effect of CBP21 on chitinaseefficiency; the chitinase is chitinase C (ChiC), a family 18endochitinase from S. marcescens (data from Vaaje-Kolstad et al., 2005,J. Biol. Chem. 280: 28492). FIG. 1D shows the crystal structure of CBP21(Vaaje-Kolstad et al., 2005, J. Biol. Chem. 280: 11313). The side chainsof the conserved histidine residues on the flat binding surface areshown in stick representation. FIG. 1E shows the crystal structure of aGH61E from Thielavia terrestris (Harris et al., 2010, Biochemistry 49:3305). The side chains of the conserved histidine residues on thebinding surface are shown in stick representation. FIG. 1F shows adetailed view of the conserved arrangement of the two histidines and theN-terminal amino group in CBP21 (dark grey) and GH61E (light grey),superimposed using PyMol. FIGS. 1D, 1E, and 1F were generated usingPyMol.

FIG. 2A shows a MALDI-TOF MS spectrum showing oxidized solublechitooligosaccharides generated by CBP21 acting on beta-chitin whiskers.This represents the very first time that such oxidized products weredetected. FIG. 2B shows a chitin chain with a GlcNAcA unit. FIG. 2Cshows 2.0 mg/mL beta-chitin treated with 1.0 μM CBP21 in the presence(left vial) or absence (right vial) of 1.0 mM ascorbic acid. After theincubation with or without ascorbic acid, the tubes were treated in anidentical manner: They were shaken and then left for one minute for thechitin material to settle/sediment. The figure shows that the disruptedmaterial in the left vial sediments much more slowly. This figure showsthe enormous potential of CBP21 in disrupting crystalline chitin whenapplied under optimal conditions, i.e., in the presence of a reducingagent and a divalent metal ion. FIG. 2D shows a MALDI-TOF MS spectrum ofproducts obtained after incubating beta-chitin (2.0 mg/mL) with 1.0 μMAnCDA (an overexpressed and purified chitin deacetylase from Aspergillusnidulans), 1.0 μM CBP21 and 1.0 mM ascorbic acid for 16 hours, showingoxidized deacetylated chitooligosaccharides (all major products containtwo acetylated sugars). A control reaction without CBP21 did not yieldsoluble products (results not shown). FIG. 2E shows a MALDI-TOF MSspectrum of products obtained after incubating beta-chitin (2.0 mg/mL)with 1.0 μM AnCDA and 0.5 μM ChiC for 8 hours, showing deacetylatedchitooligosaccharides (all major products contain one acetylated sugar).MS peaks are labelled by observed atomic mass and the degree ofpolymerization (DP) of the oligosaccharide. Labels always refer to thepeak of highest intensity in the respective cluster (which comprisesseveral adducts; see FIGS. 3A-3D). “ox” indicates the presence of aGlcNAcA at the chain end opposite to the non-reducing end. Asterisksindicate a deacetylated product. 100% relative intensity represents2.5×10⁴, 1.1×10³ and 5.4×10⁴ arbitrary units (a.u.), in FIGS. 2A, 2D,and 2E, respectively. See FIGS. 3A-3D for additional experiments forproduct verification.

FIGS. 3A, 3B, 3C, and 3D show controls of product identities byMALDI-TOF. FIG. 3A shows GlcNAcA containing products of CBP21 degradedbeta-chitin are in equilibrium with the 1,5 δ-lactone and at low pH thisequilibrium should become observable (Pocker & Green, 1973, J. Am. Chem.Soc. 95: 113). A sample with hexameric products was adjusted to pH˜3.0with acetic acid and incubated for four hours at room temperature. Thesample was then analyzed by MALDI-TOF MS. The spectrum clearly showsboth the acid form and the 1,5 δ-lactone which differ by 18 atomic massunits (amu), representing a water molecule. The equilibrium between thetwo forms will eventually lead to exchange of both oxygens added duringthe reaction with oxygens from solvent water. Indeed when productsamples obtained after carrying out the reaction in H₂ ¹⁸O (seeExamples) were adjusted to low pH, we observed not only a lactone and anacid showing a mass increase of 2 amu compared to reactions performed inH₂ ¹⁶O, but also, after some time, an acid showing a mass increase of 4amu (results not shown). FIG. 3B shows details of the mass spectrum forDP6_(ox). The fact that the mass difference between Na⁺ and K⁺corresponds to the mass of an oxygen atom could complicateinterpretation. However, the similar ratios between the single sodiumand potassium adducts and their corresponding sodium salt ofsodium/potassium adduct (often observed for sugar acids) indicate thatthe product indeed is an oxidized hexamer. A second verification of theidentity of the products observed by MALDI-TOF MS was done by addingLiCl to a final concentration of 33 mM to the sample before MALDI-TOF MSanalysis to generate new diagnostic adducts. FIG. 3C shows productscorresponding to the Li-adduct of the acid and the Li-adduct of theLi-salt of the acid were observed, thus confirming the identity of Na-and K-adducts observed in FIG. 3B. Peaks in all MALDI-TOF MS spectra arelabelled according to their observed atomic mass and the degree ofpolymerization (DP) of the oligosaccharide. “ox” stands for oxidized.FIG. 3D shows superposed PSD spectra for the hexamer fraction obtainedupon CBP21 treatment of beta-chitin in H₂ ¹⁶O (black) and H₂ ¹⁸O (grey).Inserts provide detailed views of parts of the spectrum. The data showthat the Y-ions differ by 2 amu, whereas the B ions show identicalmasses regardless of the type of water used. The data are fullycompatible with the chemical structure shown above the mass spectrum andshow that the heavy oxygen atom is introduced at the oxidized reducingend. Note that the chemical structure shown has been simplified for thepurpose of clarity: most hydroxyl groups as well as C6 are lacking. 100%relative intensity represents 4.6×10⁴, 1.1×10⁴, 0.8×10³ and 1.8×10⁴ a.u.in FIGS. 3A, 3B, 3C, and 3D, respectively.

FIG. 4 shows the effect of ascorbic acid (a reductant) on thepotentiating effect of CBP21 on chitinase efficiency. The efficiency ofbeta-chitin (0.1 mg/mL) degradation by 0.5 μM ChiC was analyzed in thepresence (top grey line on filled triangles) or absence (bottom greyline on filled diamonds) of 1.0 μM CBP21 and 1.0 mM ascorbic acid at pH8.0. A parallel reaction containing beta-chitin, ChiC and CBP21 (sameconcentrations as above) but no ascorbic acid was run as a control(central dark grey line on filled squares). The results show that CBP21promotes chitin degradation by ChiC considerably better in the presenceof ascorbic acid (all chitin is degraded after 2.5 hours), compared toconditions where ascorbic acid is absent. A parallel reaction containingbeta-chitin and ChiC (same concentrations as above), in the absence ofascorbic acid, would yield a result similar to that represented by thegrey line on filled diamonds (results not shown, but the experiment isincluded and shown in FIG. 10B), meaning that ascorbic acid alone had noeffect on ChiC efficiency.

FIGS. 5A and 5B show the analysis of soluble reaction products fromCBP21 catalysis. FIG. 5A shows after 4 days of incubation of 2 mg/mLbeta-chitin with 1.0 μM CBP21 and 1.0 mM ascorbic acid at pH 8.0,soluble products are dominated by even numbered short oligosaccharideswith acidic reducing ends. Due to the low solubility of chitinoligosaccharides longer oligosaccharides are not observed (see Examplesfor the detection of longer products). FIG. 5B shows each acidicoligosaccharide species was observed as H-, Na- and K-adducts and Na-and K-adducts of the acidic oligosaccharide Na-salt. 100% relativeintensity represents 3.5×10³ a.u.

FIGS. 6A and 6B show UHPLC separation of oxidized oligosaccharides withacidic (GlcNAcA) ends. A semi-quantitative analysis of soluble productsgenerated by CBP21 was performed by separating the oxidizedoligosaccharides by UHPLC. FIG. 6A shows a typical product spectrumshowing the periodicity of even and odd numbered products that was alsoobserved in the MALDI-TOF MS analysis (e.g., FIG. 2D and FIGS. 5A and5B). Note that the alpha- and beta-anomers of non-oxidizedoligosaccharides would be separated under these chromatographicconditions. The fact that only single peaks are observed confirms themodification of the reducing end. Peak identities were determined usingMALDI-TOF MS (results not shown). FIG. 6B shows initial reactionvelocities of CBP21 mediated chitin solubilization into hexamericproducts (full lines) or pentameric products (dashed lines) in thepresence of 5.0 (grey lines, circular data points), 1.0 (dark greylines, square data points) and 0.2 mM (black lines, triangular datapoints) ascorbic acid. These results show that the velocity of thereaction depends on the ascorbic acid concentration and thatodd-numbered products are generated at a slower rate than even-numberedproducts.

FIGS. 7A and 7B show MALDI-TOF MS analysis of products detected aftertreating 2.0 mg/mL beta-chitin with 1.0 μM CBP21 and 1.0 mM ascorbicacid in Tris buffered H₂ ¹⁸O pH 8.0 (FIG. 7A) or in Tris buffered H₂ ¹⁶OpH 8.0 saturated with ¹⁸O₂ (FIG. 7B). All major products show a massincrease of 2 amu compared to reactions performed in solutions notcontaining isotope labeled water or molecular oxygen (see FIGS. 2A, 5A,and 5 5B). FIG. 7C shows adducts of the oxidized hexameric product shownin FIG. 7B. Note the small amount of non-isotope labeled product(indicated by the arrow), most likely resulting from the initial stageof the reaction where ¹⁶O₂ was still present (prior to ¹⁸O₂ saturation;see Materials and Methods). 100% relative intensity represents 6.8×10³and 2.0×10³ a.u. in FIGS. 7A, 7B, and 7C, respectively. FIG. 7D shows ascheme for the enzymatic reaction catalyzed by CBP21. In the finaloxidized product, one oxygen comes from molecular oxygen and one fromwater.

FIGS. 8A, 8B, 8C, and 8D show the effect of various potentiallyinhibiting factors on CBP21 activity. Sodium dithionite (SD) is a wellknown oxygen scavenger routinely used to create a oxygen free(anaerobic) environments for enzyme reactions. FIG. 8A shows a MALDI-TOFMS spectrum of a reaction mixture containing 2.0 mg/mL beta-chitin, 1.0μM CBP21, 1.0 mM ascorbic acid and 10 mM sodium dithionite, incubatedfor 16 hours at 37° C. under anaerobic conditions (all vials weredegassed with a Schlenk line). The spectrum shows baseline noise and nopeaks of noteworthy intensity; in other words, there are no detectableamounts of CBP21 generated products. 100% relative intensity represents0.9×10² a.u. FIG. 8B shows that the potentiating effect of CBP21 onchitinase (ChiC) is abolished by adding sodium dithionite (SD). Reactionconditions: 0.1 mg/mL beta-chitin, 0.1 μM ChiC, 1.0 mM ascorbic acid, 10mM sodium dithionite, 1.0 μM CBP21, incubated for 16 hours at 37° C.under anaerobic conditions (all vials were degassed with a Schlenkline). End product quantities were analyzed by HPLC. FIG. 8C showsadditional control experiments, showing product development duringincubation of 0.45 mg/mL beta-chitin with 0.5 μM ChiC and 1 mM reducedglutathione (which has the same effect as ascorbic acid; see FIG. 11) in20 mM Tris-HCl, pH 8.0. Further additions/conditions were 1.0 M CBP21(standard conditions for maximum activity; dark line on filleddiamonds), 1.0 μM CBP21 in a solution with reduced oxygen concentrationobtained by gas exchange (line on plusses; note that the resultsdisplayed in FIG. 7C, showing the presence of ¹⁶O₂ even after extensivegas exchange in the Schlenk line, show that truly anaerobic conditionswere not obtained), 1.0 μM CBP21 and 2 mM potassium cyanide, a wellknown O₂ mimic (line on filled squares) and no CBP21, no furtheradditions (standard control experiment; line on filled circles).Addition of 2.0 mM sodium azide, a known inhibitor of heam proteins, didnot inhibit CBP21 activity and the curves were similar to the curve onfilled diamonds. CBP21 was also fully inhibited by oxyrace (OxyraceInc., Mansfield, Ohio), a bacterial oxidase that uses lactate ashydrogen donor in order to create an anaerobic environment (results notshown). FIG. 8D shows the production of GlcNAc₃GlcNAcA (i.e., anoxidized tetramer) during a reaction of 0.45 mg/mL beta-chitin, 1.0 μMCBP21 and 1.0 mM reduced glutathione in 20 mM Tris-HCl pH 8.0 in theabsence (line on filled diamonds) or presence (line on filled squares)of 2.0 mM potassium cyanide. This result shows that cyanide inhibits theoxidation reaction. Data shown in FIGS. 8B, 8C, and 8D are mean+/−SD(N=3); error bars (not visible for every point) indicate SD.

FIG. 9 shows the effect of divalent cations on CBP21 activity. Thefigure shows ChiC activity, monitored by measuring the concentration ofproduced (GlcNAc)₂ by HPLC. Reaction mixtures contained 2.0 mg/mLbeta-chitin in 1.0 mM ascorbic acid, 5 mM EDTA, 0.5 μM ChiC in 20 mMTris pH 8.0, in the presence or absence of 1.0 μM metal-free CBP21.Solid lines indicate reactions with metal-free CBP21; dashed linesindicate reactions without CBP21. Half way through the reaction(indicated by the arrow) MgCl₂ (final concentration 25 mM), ZnCl₂ (25mM) or buffer was added to one of two (parallel) reaction mixtures.Squares, MgCl₂ added; circles, ZnCl₂ added; triangles, buffer added. Theresults clearly demonstrate that divalent cations are essential for thefunction of CBP21; the controls show that the cations do not affect ChiCactivity.

FIGS. 10A and 10B show the importance of the conserved His114.CBP21-like and GH61 proteins contain two highly conserved histidines(H28 and H114 in CBP21), one of which is the N-terminal histidine of thesecreted mature protein. In order to probe the importance of His114 theH114A mutant variant of CBP21 was analyzed. FIG. 10A shows thatincubation of 1.0 μM CBP21^(H114A) with 2.0 mg/mL beta-chitin, 1.0 mMascorbic acid in 20 mM Tris, pH 8.0 for 16 hours at 37° C. did not leadto production of soluble oxidized oligosaccharides. 100% relativeintensity represents 3.5×10⁴ a.u. FIG. 10B shows product release upondegradation of 0.1 mg/mL beta-chitin with 0.5 μM ChiC in 1.0 mM ascorbicacid, 20 mM Tris, pH 8.0, in the presence or absence of CBP21^(WT) (topgrey line on filled triangles and dark grey line on filled diamonds,respectively) and in the presence of CBP21^(H114A) (grey line on filledsquares). The dark grey lines on filled circles show a reaction withoutCBP21 and without ascorbic acid. The data clearly show that His114 isessential for the function of CBP21 as CBP21^(H114A) is unable to boostchitinase activity, even in the presence of ascorbic acid.

FIGS. 11A and 11B show the effect of other reductants on CBP21 activity.Reaction products obtained upon incubating 2.0 mg/mL beta-chitin for 16hours at 37° C. in the presence of 1.0 μM CBP21 and (FIG. 11A) 1.0 mMreduced glutathione or (FIG. 11B) in 1.0 mM Fe(II)SO₄, in 20 mM Tris pH8.0. Note that the observed product profiles are highly similar to thoseobtained in the presence of ascorbic acid (FIGS. 5A and 5B). FIGS. 5 5Aand 5B and 6 FIGS. 6A and 6B show that the activity of CBP21 isincreased in the presence of ascorbic acid. Reductants such as reducedglutathione and Fe(II)SO₄ had similar effects on the kinetics of thedegradation reaction (results not shown). 100% relative intensityrepresents 1.5×10⁴ and 0.8×10³ a.u. in FIGS. 11A and 11B, respectively.

FIG. 12 shows the effect of ascorbic acid on beta-chitin. It is wellestablished that reductants undergo autooxidation in oxygenatedsolutions, thus generating reactive oxygen species that may putativelyoxidize biomolecules present in the surrounding medium. The figure showsMALDI-TOF MS analysis of a reaction mixture obtained after incubating asample containing 2.0 mg/mL beta-chitin and 1 mM ascorbic acid in 20 mMTris pH 8.0 for four days at 37° C. The spectrum does not show any signof soluble oxidized oligosaccharides. 100% relative intensity represents4.4×10⁴ a.u.

FIGS. 13A, 13B, 13C, and 13D show the effect of CBP21 on soluble nativeoligosaccharides. Earlier studies (Vaaje-Kolstad et al., 2005, J. Biol.Chem. 280: 11313; Suzuki et al., 1998, Biosci. Biotechnol. Biochem. 62:128) have shown that CBP21 binds specifically to beta-chitin and not tosoluble or amorphous forms of chitin. With the results presented here inmind, the ability of CBP21 to oxidize and/or hydrolyze nativeoligosaccharides of chitin was investigated. A solution containing 100μM GlcNAc₆ in 20 mM Tris pH 8.0 was incubated for 16 hours in thepresence of buffer only, 1.0 mM ascorbic acid (AA) or 1.0 mM ascorbicacid and 1.0 μM CBP21 and reaction products were analyzed by MALDI-TOFMS (FIGS. 13A, 13B, and 13C, respectively) and quantified by HPLC (FIG.13D). No degradation or oxidation of GlcNAc₆ was observed. Similarexperiments were performed with the soluble polymeric chitin-derivativechitosan; again, no degradation or oxidation products could be detected(results not shown). 100% relative intensity represents 5.7×10³, 1.4×10⁴and 4.0×10⁴ a.u. for FIGS. 13A, 13B, and 13C, respectively.

FIG. 14 shows the possible effect of Fenton chemistry on beta-chitinsolubilization. It could be envisaged that the observed CBP21 mediatedoxidohydrolytic cleavage of beta-chitin is related to generation ofconditions leading to Fenton type chemistry (analogous to what has beenproposed for cellulose degradation by cellobiose dehydrogenases (Hammelet al., 2002, Enzyme. Microb. Tech. 30: 445; Henriksson et al., 2000, J.Biotechnol. 78: 93; Hyde & Wood, 1997, Microbiol-Uk 143: 259). Although,Fenton chemistry is not particularly effective at the pH used in thisstudy (8.0), we did check whether Fenton-like processes might occur.Fenton chemistry is well known to depolymerize polysaccharides and couldyield oxidized products similar to those generated by CBP21. 2.0 mg/mLbeta-chitin was incubated for 16 hours with 0.03% H₂O₂ and 5.0 mMFe(II)SO₄ in 20 mM Tris pH 8.0, at 37° C. The soluble phase of thereaction was analyzed by MALDI-TOF MS. The mass spectrum shown in thefigure did not reveal formation of soluble products. As controls,MALDI-TOF experiments were repeated using various dilutions of theoriginal sample; samples from reactions where the H₂O₂ concentration was0.3% or 0.003% were also analyzed. Soluble products were never observed(results not shown). 100% relative intensity represents 1.2×10⁴ a.u.

FIG. 15A shows crystals of EfCBM33 (Uniprot ID:Q838S1; EF0362;uniprot.org/uniprot/Q838S1), obtained by hanging drop vapor diffusionexperiments that have been used to collect 0.95 Å data. FIG. 15B showsthe side chain of the solvent exposed Trp modeled in the 2Fo-Fc map.FIG. 15C shows superposition of the main chains of CBP21 and EfCBM33;the side chains of the conserved histidines and a surface exposedaromatic amino acid (Tyr in CBP21, Trp in EfCBM33) are shown. FIG. 15Dshows a MALDI-TOF MS spectrum of soluble products obtained aftertreating 2.0 mg/ml beta-chitin with “washed” EfCBM33 crystals dissolvedin 20 mM Tris pH 8.0; as for CBP21, the product spectrum is dominated byeven-numbered oxidized chitooligosaccharides, clearly demonstratingoxidohydrolytic activity. FIG. 15E shows n degradation of 2.0 mg/mlalpha-chitin (from shrimp shells) by 0.3 μM of the chitinase fromEnterococcus feacalis (protein name (EF0361) in the presence or absenceof 0.3 μM EfCBM33 and 1.0 mM reductant (R: reduced glutathione)incubated at 37° C. with agitation at 900 rpm. A boost of the chitinaseactivity is clearly observed in the presence of EfCBM33 and reductant.

FIGS. 16A and 16B show the speed and extent of oxidative cleavage byCBP21 activity. FIG. 16A shows the production of non-oxidized dimers(top line with diamonds), oxidized trimers (line on triangles) andoxidized tetramers (obscured line on squares) produced during incubationof 0.45 mg/mL beta-chitin, 1.0 μM CBP21, 0.5 μM ChiC and 1.0 mM reducedglutathione in 20 mM Tris-HCl pH 8.0 (hydrolysis of oxidizedoligosaccharides by ChiC produces only minimal amounts of oxidizeddimers; the three products shown represent the large majority ofoligomers produced under these conditions). After five hours,approximately 4.9% of the total sugars (theoretical number based onchitin concentration) are oxidized*. FIG. 16B shows production ofoxidized sugars during incubation of 0.45 mg/mL beta-chitin, 1.0 μMCBP21 and 1.0 mM reduced glutathione in 20 mM Tris-HCl pH 8.0. Thedegree of oxidation was determined by rapid conversion of the treatedchitin with a large dose of a chitinase cocktail followed by UHPLCdetection of oxidized dimers (filled diamonds) and trimers (filledtriangles), as described in the Materials and Methods section. Thelinear part of the reaction represents an oxidation rate ofapproximately 1 per minute**. Maximum levels are reached after 2-3 hoursand represent a degree of oxidation of 7.6% of the total sugars(theoretical number based on chitin concentration)***. Data in bothpanels are mean+/−SD (N=3); error bars (not visible for every point)indicate SD.

*The end concentrations of oxidized trimer and tetramer are 53 and 55μM, respectively, giving rise to a total of 108 μM GlcNAcA. The molarconcentration of GlcNAc in the solution is 2217 μM. Thus the degree ofoxidation is 108/2217; in other words 4.9% of the sugars are GlcNAcA.**The rates calculated for oxidized dimer and oxidized trimer are 0.68and 0.60 μM/min. When added, the rate of oxidized products generated is1.28 μM/min and when taking the CBP21 concentration into account (1.0μM), the rate of oxidohydrolysis is 1.28 per minute. ***At the maximumlevels reached, CBP21 has produced 93 μM and 75 μM oxidized dimer andoxidized trimer, respectively, which adds up to 168 μM GlcNAcA. Themolar concentration of GlcNAc in the solution is 2217 μM. Thus thedegree of oxidation is 168/2217; in other words 7.6% of the sugars areGlcNAcA.

FIG. 17 shows MALDI-TOF MS analysis of soluble products generated by aCBM33 protein (1 μM CelS2) incubated with microcrystalline cellulose(2.0 mg/ml AVICEL®) in 20 mM Tris-HCl buffer pH 8.0 in the presence of1.0 mM ascorbic acid (external electron donor), incubated for 20 hours,at 50° C. with horizontal agitation at 250 rpm. The main peaks areannotated with molecular weight and degree of polymerization (DP). Allannotated peaks are Na-adducts of oxidized cellooligosaccharides (hencethe subscript “ox”). More detailed analysis of the adduct clusters isshown in FIG. 18.

FIG. 18 shows MALDI-TOF MS analysis of the adduct cluster of theoxidized cellooligosaccharide hexamer generated by CelS2 (from the samesample as described in FIG. 17 legend). Peaks are annotated withmolecular weight, degree of polymerization (DP); “ox” indicatesoxidation. The spectrum shows small quantities of the native hexamericoligosaccharide (m/z=1013.14). The native oligosaccharides may arisefrom CelS2 substrate cleavage near the reducing end or liberation due tosubstrate disruption (see also FIG. 19). The dominant peak representsthe Na-adduct of the oxidized hexamer (m/z=1029.14). Additionally, peaksrepresenting the K-adduct of the oxidized hexamer (m/z=1045.08) and theNa-adduct of the Na-salt of the oxidized hexamer (m/z=1051.13) can alsobe observed.

FIG. 19 shows HPAEC analysis of soluble products generated afterincubation of 1.0 μM CelS2 with 10 mg/ml AVICEL® in 50 mM Tris-HCl pH8.0, 1 mM MgCl₂, at 50° C., 900 rpm (horizontal agitation) in thepresence (“pink”, upper line) or absence (“dark purple”, lower line) of0.5 mM reduced gluathione (external electron donor). In the absence ofreduced glutathione only small amounts of native cellooligosaccharidesare observed (Glc3-Glc6) whereas larger amounts of both nativecellooligosaccharides and oxidized cellooligosaccharides are observedwhen reduced glutathione is present. The DP of the oxidizedcellooligosaccharides is indicated by n, where n=3 (the shortestlabelled oxidized cellooligosaccharide has a DP of three (=n)). Thepresence of small amounts of native cellooligosaccharides was also seenin the control reaction without added CelS2 (10 mg/ml AVICEL® in 50 mMTris-HCl pH 8.0 in the presence and absence of 0.5 mM reducedglutathione; results not shown). Thus, the substrate itself containssome shorter soluble non-oxidized cello-oligomers that are released uponincubation, even without CelS2. It is well known from the literaturethat AVICEL® has a relatively low degree of polymerization (Wallis etal., 1992, Carbohydrate Polymers 17: 103-110). Additionally, chaincleavage by CelS2 near the reducing end of a cellulose chain will giverise to such products. Peak annotation was performed by comparing thechromatogram with the chromatogram obtained for a chemically preparedmixture of oxidized cellooligosaccharides with lengths varying from DP1to DP10. Note the periodicity of the oxidized cellooligosaccharides alsoobserved by MALDI-TOF MS analysis (FIG. 17).

FIG. 20 shows total sugar (g/l) ([Glc]+cellobiose which is reported as[Glc]) after incubation of 1 μM CelS2 or E7 and 0.05 μl/ml CELLUCLAST™(“CC”) with 10 mg/ml filter paper for 24 or 120 hours in the presence orabsence of 1 mM reduced glutathione (“RG”). Reactions were run in 50 mMBis-tris/HCl pH 6.5, 1 mM MgCl₂, at 50° C. and 900 rpm (horizontalagitation). Control reactions containing the cellulosic substratesuspended in the same buffer as used for the enzyme assays gave nodetectable signal for either glucose or cellobiose.

FIG. 21 shows cellobiose concentration (g/l) after 18 or 40 hoursincubation of 1.0 μM CelS2 and 5 μg/ml Cel7A (“Cel7A”) with 10 mg/mlfilter paper in the presence or absence of 1 mM reduced glutathione(“RG”). These reactions were conducted in 50 mM sodium acetate pH 5.5, 1mM MgCl₂, at 50° C., 900 rpm (horizontal incubation). Only thecellobiose concentration was quantified as the amount of glucose wasminor (less than 5% of the total sugar). Control reactions containingthe cellulosic substrate suspended in the same buffer as used for theenzyme assays gave no detectable signal for either glucose orcellobiose.

FIG. 22 shows HPAEC profiles showing products obtained after incubatingCelS2 (1.0 μM) with microcrystalline cellulose (10 mg/ml AVICEL®) in 20mM Tris-HCl buffer pH 8.0 in the presence of 1.0 mM ascorbic acid(external electron donor), in the presence (line labelled “pink”, lowerline) or absence (line labelled “green” line, upper line) of 2.0 mMpotassium cyanide. Products were analyzed after incubation for 24 hoursat 50° C. with vertical agitation at 900 rpm. The two remaining (lower)lines represent the same conditions as noted above (CelS2+cyanide;“blue” line, buffer+cyanide; “brown” line), in the absence of ascorbicacid. The DP of the oxidized cellooligosaccharides is indicated (n=3),i.e., the shortest visible oxidized cellooligosaccharide has a DP ofthree (GlcNAc-GclNAc-GlcNAcA).

FIG. 23A shows MALDI-TOF MS analysis of soluble products generated by 1μM CelS2-WT and FIG. 23B shows MALDI-TOF MS analysis of soluble productsgenerated by 1 μM CelS2-H144 upon incubation with microcrystallinecellulose (10 mg/ml AVICEL®) in 20 mM Tris-HCl buffer pH 8.0 in thepresence of 1.0 mM ascorbic acid (external electron donor), for 24 hoursat 50° C. with horizontal agitation at 250 rpm. The main peaks areannotated with molecular weight and degree of polymerization (DP). Allannotated peaks are Na adducts of native cellooligosaccharides, oxidizedcellooligosaccharides (denoted “ox”) or the sodium salts of oxidizedcellooligosaccharides. Neither native nor oxidized cellooligosaccharidescan be observed for the reaction with CelS2-H144 (FIG. 23B). The lowintensity peak observed at 1199.55 m/z does not correspond to anyoligosaccharide resulting from cellulose oxidation or hydrolysis and islikely to be a background component. The presence of nativecellooligosaccharides is likely to be the result of the low mean DP ofthe substrate (AVICEL®; Wallis et al., 1992, Carbohydrate Polymers 17:103-110); chain cleavage by CelS2 near the reducing end of a cellulosechain will also give rise to such products. See legend to FIG. 19 foradditional discussion.

FIG. 24 shows the effect of CelS2 (1.0 μM) on the degradation of steamexploded saw dust from poplar (SEP; 2.0 mg/ml) by CELLUCLAST™ (0.016μl/ml) in 20 mM sodium acetate buffer pH 5.5, 1.0 mM MgCl₂, 1.0 mMreduced glutathione (RG; external electron donor) incubated for 20 hoursat 50° C., 250 rpm (horizontal agitation). The figure shows cellobioserelease after 20 hours.

FIG. 25 shows degradation of high molecular weight filter papercellulose (10 mg/ml) in 20 mM sodium acetate buffer pH 5.5 by 0.8 μg/mlCELLUCLAST™ (CC), in the presence or absence of 40 μg/ml CelS2 and 0.5mM reduced glutathione (RG) as shown by the increase of solublecellooligosaccharides (Glc and Glc₂; converted to total Glc) over time.In reactions where no CelS2 was present, 40 μg/ml purified BSA was addedin order to maintain an identical protein load. Under these conditions,reactions with only CelS2 did not yield detectable amounts of Glc orGlc₂ (not shown). The chitin-active CBM33, CBP21, did not affect CCefficiency (not shown). RG had no effect in reactions with only CC; onlyone of the two overlapping curves is shown. Data are mean+/−SD (N=3);error bars indicate SD.

FIG. 26 shows degradation of cellulose in a reaction of 10 mg/ml filterpaper with a combination of 40 μg/ml CelS2 (or 40 μg/ml BSA tocompensate for the protein load in reactions without CelS2) and 5 μg/mlpurified HjCel7A in 1 mM MgCl₂, 20 mM sodium acetate buffer, pH 5.5. Byfar the major products of these reactions are cellooligosaccharidesreleased by the cellulases (whose activity is boosted by CelS2). In thecase of HjCel7A, the main product is cellobiose and formation of thisproduct is shown. RG, indicates the presence of a reductant, in thiscase reduced glutathione. The label “+/−RG” indicates that productioncurves for Cel7A alone (no CelS2 present) with and without RG wereessentially identical. The reactions were run at 50° C.

FIG. 27 shows HPAEC profiles of products obtained after incubatingAVICEL® with the N-terminal CBM33 domain of CelS2 in the absence ofreductant (bottom line) and in the presence of reduced glutathione (2ndfrom bottom line), gallic acid (darker of upper lines) or ascorbic acid(lighter of upper lines), which all serve as external electron donor. 1μM of the N-terminal CBM33 domain of CelS2 was incubated with 10 mg/mlAVICEL® in the presence of 0.8 mM of one of the reductants, or in theabsence of any reductant. Reactions were run in 50 mM succinate bufferpH 5.5, 1 mM MgCl₂, at 50° C. and 900 rpm (vertical agitation) for 20hours. FIG. 28 shows total sugar (g/l) [Glc] (glucose and cellobiosereported as [Glc]) released after incubation of 0.08 μl/ml CELLUCLAST™(CC) in the presence or absence of 1 μM E7 and 2 mM reduced glutathione(RG) as external electron donor with 2 mg/ml filter paper cellulose.Reactions were incubated in 20 mM sodium acetate buffer pH 5.5, 1 mMMgCl₂, at 50° C. and 900 rpm (horizontal agitation) for up to 50 hours.RG had no effect in reactions with only CC; only one of the twooverlapping curves is shown. Control reactions with filter papersuspended in the same buffer as used for the enzyme assays, or E7 inabsence of cellulases did not yield detectable amounts of Glc or Glc₂(not shown).

FIG. 29 shows MALDI-TOF MS analysis of soluble products generated by 1μM E7 incubated with 10 mg/ml AVICEL® in 20 mM Tris-HCl buffer pH 8.0and 1 mM MgCl₂ in the presence of 1 mM ascorbic acid as externalelectron donor, incubated for 20 hours, at 50° C. with verticalagitation at 900 rpm. The oxidized oligosaccharides (Glc₃₋₇GlcA) areobserved as sodium adducts, and as sodium adducts of the oligosaccharidesodium salts, and annotated with their molecular weights (m/z); nativecello-oligosaccharides are also present but have been excluded fromannotation. Such native oligosaccharides might be the result of CBM33cleavage near the reducing end of the substrate.

FIG. 30 shows HPAEC profiles of products generated by 1 μM full lengthCelS2 in the presence (top grey line) or the absence (bottom dark greyline) of 1 mM ascorbic acid (AA), or by the N-terminal CBM33 domain ofCelS2 in the presence of AA (second top, dark line), or by theC-terminal CBM2 domain of CelS2 in the presence of AA (second bottom,dark line) upon incubation with 10 mg/ml AVICEL®. Products were analyzedafter 20 hours of incubation in 20 mM ammonium acetate buffer pH 5.0, 1mM MgCl₂ at 50° C. and 900 rpm (vertical agitation). The chromatogram isenlarged to emphasize the oxidized cello-oligosaccharides produced bythe full length CelS2 and the N-terminal CBM33 domain of CelS2.

FIG. 31 shows reactivation of activity by adding metals to EDTA treatedN-terminal CBM33 domain of CelS2. 0.8 mg/ml CBM33 was incubated with 400μM EDTA for 3 hours at 20° C. This EDTA treated enzyme (40 μg/ml) wasincubated with 10 mg/ml AVICEL® in 50 mM MES buffer pH 6.6 containing1.7 mM reduced glutathione and one of 6 different metal ions (10 μM;Mg²⁺, Fe³⁺, Zn²⁺, Co²⁺, Ca²⁺, Cu²⁺). The remaining concentration of EDTAin these reaction mixtures was 20 μM. The superimposed HPAECchromatograms show products released after 20 hours of incubation at 50°C. Only Cu²⁺ (top, grey line) reactivated the enzyme under theseconditions and when using these low metal concentrations.

FIG. 32A shows an alignment of GH61 sequences using ClustalW. TheHjGH61A (SEQ ID NO: 15) sequences were aligned to a profile created bystructurally aligning TtGH61E (SEQ ID NO: 1 without the leader peptide)and HjGH61B (SEQ ID NO: 16). N-terminal signal peptides present in thenatural primary gene products were removed from the sequences prior toproducing the alignment. FIG. 32B shows the structural superposition ofTtGH61E and HjGH61B. The side chains of six conserved active site andsurface residues each shaded in grey in the alignment of FIG. 32A areshown in dark grey in FIG. 32B. An insertion present in HjGH61B is shownin the shaded section in FIG. 32A and in the 3 turn helix in FIG. 32B.

FIG. 33A shows the structure of CBP21. FIG. 33B shows a comparison ofthe structure of CBP21 with a structural model of E7. FIG. 33C shows acomparison of the structure of CBP21 with the structure of TtGH61E Notethe similarity between E7 and TtGH61E (Met and Val at positions 207 and161 respectively are both hydrophobic residues; the correspondingresidue in CBP21, T183, is more polar). CBP21 lacks a histidine (CBP21has D182), a tyrosine (CBP21 has F187) and a glutamine (CBP21 has E60)that are conserved in the two GH61s shown. Residues are numbered as theyappear in the primary gene product, i.e., a protein with an N-terminalsignal peptide. Mature correctly processed proteins start with ahistidine, which would then be histidine 1 (such as shown in thealignment of FIG. 32A). H28, H37 and H19 correspond to this histidine 1in FIGS. 33A, 33B, and 33C, respectively.

FIG. 34 shows HPAEC chromatograms for production of oligosaccharidesfrom microcrystalline cellulose (AVICEL®) by TtGH61E (upper curve, withhigher peaks) and TaGH61A (lower curve). GH61 proteins (140 μg/ml) wereincubated at 50° C. with 10 mg/ml AVICEL® in 50 mM MES buffer pH 6.6containing 2.4 mM ascorbic acid (a reductant) for 24 hours withhorizontal agitation at 700 rpm.

FIG. 35 shows chromatograms (Rezex RFQ-Fast Fruit H+ column) forproduction of glucose from AVICEL® and Filter Paper by CELLIC™ CTec2(final concentration of added protein was 1.1 μg/mL) in the presence orabsence of ascorbic acid. The enzymes were incubated at 50° C. with 10mg/ml substrate (AVICEL® or filter paper) in 50 mM MES buffer pH 6.6with or without 0.5 mM ascorbic acid for 24 hours with horizontalagitation at 700 rpm.

DETAILED DESCRIPTION OF THE INVENTION

Surprisingly, the inventors have now found that CBM33 and GH61 proteinsas oxidohydrolases exploit molecular oxygen and water to introduce chainbreaks on the surfaces of crystalline polysaccharides, i.e., on thesurface of a solid phase, to open up the inaccessible polysaccharidematerial for hydrolysis by normal glycoside hydrolases. Although notwishing to be bound by theory it is believed that the carbohydrate chainis oxidized by molecular oxygen and chain cleavage is accomplished by aconcomitant hydrolysis (FIG. 7D). In view of this mechanism we refer tothe enzymes as oxidohydrolases. However, these enzymes may alternativelybe referred to herein as polysaccharide degrading enzymes (or simplyenzymes) in view of their ability to effect or assist cleavage ofglycosidic bonds in polysaccharides. Enzymatic cleavage of crystallinepolysaccharides in a reaction dependent on reactive oxygen species hasso far not been described.

These enzymes have flat surfaces that bind to the flat, solid,well-ordered surfaces of crystalline material and catalyze chain breaks.The chain break will result in disruption of crystalline packing andincreased substrate accessibility, an effect that may be augmented bythe modification of one of the new chain ends. At the cleavage point oneof the new ends is a normal non-reducing end (indicated by R—OH in FIG.7D). The other new end would have been a new reducing end if thecleavage had been performed by a normal glycoside hydrolase. However, inthis case the product is different and the last sugar is oxidized tobecome 2-(acetylamino)-2-deoxy-D-gluconic acid (FIG. 7D). This novel“acidic chain end” will interfere with normal crystal packing because itwill not have the normal chair conformation of the sugar ring andbecause it carries a charge. Effects on non-crystalline substrates arealso possible.

Genes encoding these oxidohydrolases (such as genes encoding members ofthe CBM33 or GH61 families) are abundant in chitin- andcellulose-degrading microorganisms. As assessed by gene sequences, CBM33and GH61 proteins are found both as single-domain proteins (i.e.,consisting of a CBM33 or GH61 domain only) and as multi-domain proteins(i.e., consisting at least one more domain, often a domain that isputatively involved in substrate binding). When the CBM33 or GH61 domaincontaining proteins have more than one domain, the additional domainsare usually coupled to the C-terminus of the CBM33 or GH61 domainbecause the N-terminus of the CBM33 or GH61 domain is essential foroxidohydrolytic activity. Knowledge of the mode of action of theseenzymes has allowed their catalytic efficiency to be optimized toachieve more efficient enzymatic conversion of biomass into sugars whichmay be used for fermentation.

In view of the identification of the role of molecular oxygen incatalysis, it has now been found that the efficiency of the reaction canbe improved by the addition of reductants that can act as electron donorand/or generate reactive oxygen species. In the presence of divalentmetal ions reductants improve enzymatic conversion of recalcitrantpolysaccharides.

Thus, in a first aspect, the present invention provides a method ofdegrading or hydrolyzing a polysaccharide comprising contacting saidpolysaccharide with one or more oxidohydrolytic enzymes, wherein saiddegradation or hydrolysis is carried out in the presence of at least onereducing agent and at least one divalent metal ion.

As referred to herein “degrading” said polysaccharide refers todegradation by disruption of the glycosidic bonds connecting the sugarmonomers in the polysaccharide polymer.

The degradation of said polysaccharide is enhanced by the use of saidreducing agents and metals relative to performance of said methodwithout those means, thus the rate or degree of disruption of theglycosidic bonds that connect the sugar monomers is increased. This mayreadily be determined by measuring the product formation, e.g., atcertain defined time points or by measuring the amount of undegradedpolysaccharide substrate which remains, e.g., at certain defined timepoints. This can be carried out using methods that are well known in theart, based on, e.g., determination of liberated reducing sugars (Horn etal., 2004, Carbohydrate Polymers 56(1): 35-39 and references therein) ordetermination of liberated fragments, e.g., cellulose or chitinfragments, e.g., by quantitative analysis of chromatograms obtained uponHigh Performance Liquid Chromatography (Hoell et al., 2005, Biochim.Biophys. Acta 1748(2): 180-190). Preferably said measure of degradationis assessed in the presence of one or more relevant saccharolyticenzymes as described hereinafter.

If the rate of degradation (e.g., hydrolysis), i.e., the number of bondsdisrupted (e.g., hydrolyzed) in a certain time period is greater whenthe substrate has been exposed to the oxidohydrolytic enzyme in thepresence rather than absence of reducing agents and metal ions, then therate of degradation is considered to be enhanced. Preferably the use ofreducing agents and metal ions reduces the time taken for degradation(either complete or to the same level of partial degradation, e.g., whenadditional saccharolytic enzymes are used, see hereinafter) by at least2, 3, 4, 5, 6, 7, 8, 9 or 10 fold. Alternatively expressed, the use ofreducing agents and metal ions increases the rate of degradation by atleast 2, 3, 4, 5, 6, 7, 8, 9 or 10 fold.

“Hydrolyzing” refers to the chemical reaction in which water reacts witha compound to produce other compounds and involves the splitting of abond and the addition of the hydrogen cation and the hydroxide anionfrom the water. In the case of hydrolysis of polysaccharides glycosidicbonds are cleaved by hydrolysis. The hydrolysis of polysaccharides tosoluble sugars is referred to as “saccharification”. Hydrolysis ofpolysaccharides as referred to herein results in degradation of thepolysaccharide into smaller polysaccharides, including oligosaccharidesand saccharide monomers such as glucose.

Hydrolysis of the polysaccharide may be partial or complete. In the caseof complete hydrolysis, complete saccharification is achieved, i.e.,only soluble sugars (e.g., mono- and di-saccharides) remain. In partialhydrolysis, in addition to soluble sugars, larger oligosaccharides andpolysaccharides remain. As described herein methods of the inventioninclude methods in which only oxidohydrolytic enzymes are used fordegradation or in which both oxidohydrolytic enzymes and saccharolyticenzymes are used for degradation. In the former case, preferably atleast 0.05-10%, e.g., 0.05 to 5%, preferably 0.1 to 1% of the glycosidicbonds of the starting polysaccharide are degraded (i.e., disrupted,e.g., hydrolyzed) into oligosaccharides which may be separate from thepolysaccharide substrate or may remain associated despite cleavage. Inthe latter case in which saccharolytic enzymes are also used, preferablyat least 50% (especially preferably 60, 70, 80, 90, 95, 96, 97, 98, 99or 100%) of the glycosidic bonds of the starting polysaccharide aredegraded, e.g., hydrolyzed. Alternatively expressed, in the latter case,preferably at least 50% (especially preferably 60, 70, 80, 90, 95, 96,97, 98, 99 or 100%) of the starting polysaccharide is hydrolyzed intomono- or di-saccharides.

In relation to cellulose, the level of degradation may be assessed bydetermining the increase in the level of cellobiose and/or glucose.

As referred to herein said “polysaccharide” is a polymeric carbohydratestructure, formed of repeating units (either mono- or di-saccharides)joined together by glycosidic bonds and having the general formula(C₆H₁₀O₅)_(n), e.g., in which 40≤n≤3000. Preferably said polysaccharideis at least partially crystalline, i.e., is in a crystalline form or hascrystalline portions, i.e., a form or portion which shows a repeating,three-dimensional pattern of atoms, ions or molecules having fixeddistances between the constituent parts.

Preferably said polysaccharide is cellulose, hemicellulose or chitin andmay be in isolated form or may be present in impure form, e.g., in acellulose-, hemicellulose- or chitin-containing material (i.e., apolysaccharide-containing material), which optionally may contain otherpolysaccharides, e.g., in the case of cellulose, hemicellulose and/orpectin may also be present.

By way of example, the cellulose-containing material may be stems,leaves, hulls, husks and cobs of plants or leaves, branches and wood oftrees. The cellulose-containing material can be, but is not limited to,herbaceous material, agricultural residues, forestry residues, municipalsolid wastes, waste paper and pulp and paper mill residues. Thecellulose-containing material can be any type of biomass including, butnot limited to, wood resources, municipal solid waste, wastepaper, cropsand crop residues (see, for example, Wiselogel et al., 1995, in“Handbook on Bioethanol” (Charles E. Wyman, editor), pp. 105-118).Preferably the cellulose-containing material is in the form oflignocellulose, e.g., a plant cell wall material containing lignin,cellulose and hemicellulose in a mixed matrix.

In a preferred aspect, the cellulose-containing material is corn stover.In another preferred aspect, the cellulose-containing material is cornfiber, corn cobs, switch grass or rice straw. In another preferredaspect, the cellulose-containing material is paper and pulp processingwaste. In another preferred aspect, the cellulose-containing material iswoody or herbaceous plants. In another preferred aspect, thecellulose-containing material is bagasse.

“Cellulose” is a polymer of the simple sugar glucose covalently bondedby beta-1,4-linkages. Cellulose is a straight chain polymer: unlikestarch, no coiling or branching occurs and the molecule adopts anextended and rather stiff rod-like conformation, aided by the equatorialconformation of the glucose residues. The multiple hydroxyl groups onthe glucose from one chain form hydrogen bonds with oxygen molecules onthe same or on a neighbour chain, holding the chains firmly togetherside-by-side and forming microfibrils with high tensile strength.

Compared to starch, cellulose is also much more crystalline. Whereasstarch undergoes a crystalline to amorphous transition when heatedbeyond 60-70° C. in water (as in cooking), cellulose requires atemperature of 320° C. and pressure of 25 MPa to become amorphous inwater.

Several different crystalline structures of cellulose are known,corresponding to the location of hydrogen bonds between and withinstrands. Natural cellulose is cellulose I, with structures I_(α) andI_(β). Cellulose produced by bacteria and algae is enriched in I_(α)while cellulose of higher plants consists mainly of I_(β). Cellulose inregenerated cellulose fibers is cellulose II. The conversion ofcellulose I to cellulose II is not reversible, suggesting that celluloseI is metastable and cellulose II is stable. With various chemicaltreatments it is possible to produce the structures cellulose III andcellulose IV.

“Hemicellulose” is derived from several sugars in addition to glucose,especially xylose but also including mannose, galactose, rhamnose andarabinose. Hemicellulose consists of shorter chains than cellulose;around 200 sugar units. Furthermore, hemicellulose is branched, whereascellulose is unbranched.

“Chitin” is defined herein as any polymer containing beta-(1-4) linkedN-acetylglucosamine residues that are linked in a linear fashion.Crystalline chitin in the alpha form (where the chains runanti-parallel), beta form (where the chains run parallel) or gamma form(where there is a mixture of parallel and antiparallel chains),amorphous chitin, colloidal chitin, chitin forms in which part (e.g., upto 5, 10, 15 or 20%) of the N-acetylglucosamine sugars are deacetylatedare all included within the definition of this term.

Other forms of chitin that are found in nature include copolymers withproteins and these copolymers, which include protein chitin matricesthat are found in insect and crustacean shells and any other naturallyoccurring or synthetic copolymers comprising chitin molecules as definedherein, are also included within the definition of “chitin”.

The term “chitin” thus includes purified crystalline alpha, beta andgamma preparations, or chitin obtained or prepared from natural sources,or chitin that is present in natural sources. Examples of such naturalsources include squid pen, shrimp shells, crab shells, insect cuticlesand fungal cell walls. Examples of commercially available chitins arethose available from sources such as France Chitin, Hov-Bio, Sigma,Sekagaku Corp, amongst others.

As referred to herein “contacting” said polysaccharide with anoxidohydrolytic enzyme refers to bringing the two entities together inan appropriate manner to allow the catalytic properties of the enzyme tobe effective.

The precise kinetics of the reaction between the oxidohydrolytic enzymeand the polysaccharide will depend on many factors, such as the type ofpolysaccharide to be degraded, the amount of enzyme present, thetemperature and the pH. The type of polysaccharide and its degree ofamorphousness will vary with the substrate source andisolation/purification process, but can be assessed, for example, bymeasuring the degree of crystallinity of the substrate (which is amethod known in the art).

Taking these considerations into account one can determine appropriateincubation times and conditions to maximize degradation (e.g.,hydrolysis with glycoside hydrolases). Exemplary methods are discussedbelow.

Thus, the polysaccharide and oxidohydrolytic enzyme are mixed togetheror contacted with one another to allow their interaction. This maysimply involve directly mixing solutions of the different components orapplying the enzyme to the polysaccharide-containing material.

As referred to herein “one or more” preferably denotes 2, 3, 4, 5 or 6or more of the recited enzymes. When more than one of the enzymes isused they may be selected in line with the substrate to be used, e.g.,to provide complementary or synergistic action. Thus, for example,oxidohydrolytic enzymes may be combined which are effective on differentregions of the substrate, e.g., different crystal faces. Preferredcombinations are described hereinafter.

As used herein an “oxidohydrolytic enzyme” is an enzyme which usesmolecular oxygen or an activated form thereof (“reactive oxygenspecies”) for cleavage of glucoside bonds in polysaccharides, preferablychitin or cellulose. The newly generated chain ends are one normalnon-reducing end and an oxidized “acidic” end that, in the case ofchitin is a 2-(acetylamino)-2-deoxy-D-gluconic acid and in the case ofcellulose is a gluconic acid.

Preferably said enzyme has a metal binding site and requires thepresence of a divalent metal ion for full activity. The structuralenvironment of this metal ion is diagnostic (and unifying) for the CBM33and GH61 enzymes. The metal is bound by at least three ligands that arefully conserved in both families: (1) a histidine that is in position 1of the mature protein (i.e., the N-terminal residue of the protein afterthe signal peptide for secretion has been cleaved off); (2) theN-terminal amino group of the mature protein; and (3) another histidineresidue that is fully conserved.

Oxidohydrolases belonging to the CBM33 or GH61 family can be identifiedby analysis of gene sequences (and the corresponding predicted aminoacid sequences of the gene products), using standard bioinformaticmethods. For example one can use an existing multiple sequence alignmentof CBM33 or GH61 enzymes, for example represented by a Hidden MarkovModel, to search for homologous sequences in sequence databases.Sequences retrieved by such searches would be highly likely to be activeoxidohydrolases. More certainty may be obtained by (1) checking that thegene encodes a protein with a signal peptide for secretion, using, e.g.,the programme SignalP; (2) checking that the N-terminal residue aftercleavage of the signal peptide (cleavage site to be predicted using,e.g., SignalP) is a histidine; (3) checking that there is anotherhistidine in the protein sequence that aligns with a fully or almostfully (>90%) conserved histidine in the multiple sequence alignment; (4)using model-building by homology, using automated servers such asSwiss-Model, to check that this second histidine is likely to be locatedclose to the N-terminus and the N-terminal histidine.

The skilled person can readily determine by experiment whether a proteinis an oxidohydrolytic enzyme according to the above described definitionby determining if it can cleave glucoside bonds and if this processbecomes more effective in the presence of molecular oxygen, a reductantand a divalent metal ion. In addition, one may test whether the putativeoxidohydrolase works synergistically with known saccharolytic enzymepreparations and whether the magnitude of this synergistic effectdepends on the presence of reductants and divalent metal ions.Experiments such as those conducted in the examples may be used, thusthe effect of reductants and metal ions on enzymatic activity may beassessed.

Preferably said oxidohydrolytic enzyme contains at least one domain thaton the basis of sequence similarity as analyzed in, e.g., the currentPfam or CAZy databases is classified as a CBM33 or GH61 family protein.When the CBM33 or GH61 containing proteins have more than one domain,the additional domains are usually coupled to the C-terminus of theCBM33 or GH61 domain because the N-terminus of the CBM33 or GH61 domainis essential for oxidohydrolytic activity (see below). The CBM33 familyhas been classified by the CAZy (CArbohydrate-Active EnZymes) system asa carbohydrate-binding module family implying the absence of enzymaticactivity. GH61 proteins have been classified as glycoside hydrolases.However, neither classification is correct in view of the resultspresented by the inventors and clearly the CAZy classification of thesetwo protein families needs to be corrected. The CAZy classification isbased on sequence similarity, grouping protein domains that share acertain minimal level of sequence similarity into one family. The CBM33and GH61 domains share similar functions and they share a similarstructural fold, the core of which being a twisted beta-sheet sandwichlike fold, similar to that seen for fibronectin type-III domains (FIGS.1D and 1E). They also share a fully conserved, diagnosticsurface-located structural element (FIG. 1F) consisting of a histidinein position 1 of the mature protein (i.e., the protein after the signalpeptide driving secretion has been cleaved off), another fully conservedhistidine residue that is known to be crucial for catalytic activity andan N-terminal amino group that acts with these two histidines in bindinga metal ion. Despite these many unifying and diagnostic characteristics,the two families share little sequence identity and will therefore mostlikely remain in two different families even after correction of theCAZy classification.

The oxidohydrolytic enzyme is preferably a class GH61 protein. Thus theoxidohydrolytic enzyme of the invention may contain, consist or consistessentially of a GH61 domain or GH61 protein or a biologically activefragment thereof. In this context, “consists essentially of” indicatesthat additional amino acids may be present in the protein, in additionto those that make up the GH61 domain or protein. Preferably there are1-3, 1-5, 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90or 90-100 or more additional amino acids present. These additional aminoacids are in general present C terminal to the GH61 domain.

As mentioned above, the oxidohydrolytic enzyme can comprise a GH61domain or protein. Additional modules or domains may thus be present inthe protein, which, when present are preferably at the C-terminus.

In a preferred feature a native GH61 domain or protein or a biologicallyactive fragment thereof is used though variants of the native form maybe used, some of which are described hereinafter.

Oxidohydrolytic enzymes which comprise or consist of a GH61 domain orprotein or its fragments or variants are referred to herein,collectively, as GH61 proteins or GH61 family members or proteins.

Examples of suitable native proteins in this family are provided in thetable below which provides relevant database accession numbers which arehereby incorporated by reference.

Protein Name Organism GenBank Uniprot Cel1 Agaricus bisporus AAA53434.1Q00023 D649 AfA5C5.025 Aspergillus fumigatus CAF31975.1 Q6MYM8endo-beta-1,4-glucanase Aspergillus kawachii BAB62318.1 Q96WQ9 B (EglB)(Cel61A) AN1041.2 Aspergillus nidulans EAA65609.1 C8VTW9 FGSC A4AN3511.2 Aspergillus nidulans EAA59072.1 FGSC A4 AN9524.2 Aspergillusnidulans CBF83171.1 C8VI93 FGSC A4 EAA66740.1 AN7891.2 Aspergillusnidulans EAA59545.1 FGSC A4 AN6428.2 Aspergillus nidulans EAA58450.1C8V0F9 FGSC A4 AN3046.2 Aspergillus nidulans EAA63617.1 C8VIS7 FGSC A4AN3860.2 Aspergillus nidulans EAA59125.1 C8V6H2 FGSC A4endo-beta-1,4-glucanase Aspergillus nidulans ABF50850.1 (AN1602.2) FGSCA4 EAA64722.1 AN2388.2 Aspergillus nidulans EAA64499.1 C8VNP4 FGSC A4An14g02670 Aspergillus niger CBS CAK46515.1 A2R313 513.88 An15g04570Aspergillus niger CBS CAK97324.1 A2R5J9 513.88 An15g04900 Aspergillusniger CBS CAK42466.1 A2R5N0 513.88 An04g08550 Aspergillus niger CBSCAK38942.1 A2QJX0 513.88 An08g05230 Aspergillus niger CBS CAK45495.1A2QR94 513.88 An12g02540 Aspergillus niger CBS CAK41095.1 A2QYU6 513.88An12g04610 Aspergillus niger CBS CAK97151.1 A2QZE1 513.88 AO090005000531Aspergillus oryzae BAE55582.1 RIB40 AO090001000221 Aspergillus oryzaeBAE56764.1 RIB40 AO090138000004 Aspergillus oryzae BAE64395.1 RIB40AO090023000056 Aspergillus oryzae BAE58643.1 RIB40 AO090023000159Aspergillus oryzae BAE58735.1 RIB40 AO090023000787 Aspergillus oryzaeBAE59290.1 RIB40 AO090103000087 Aspergillus oryzae BAE65561.1 RIB40AO090012000090 Aspergillus oryzae BAE60320.1 RIB40 GH61A Botryosphaeriarhodina CAJ81215.1 CBS 247.96 GH61B Botryosphaeria rhodina CAJ81216.1CBS 247.96 GH61C Botryosphaeria rhodina CAJ81217.1 CBS 247.96 GH61DBotryosphaeria rhodina CAJ81218.1 CBS 247.96 Cel6 CochliobolusAAM76663.1 Q8J0H7 heterostrophus C4 unnamed protein product Coprinopsiscinerea CAG27578.1 Cel1 Cryptococcus AAC39449.1 O59899 neoformans var.neoformans CNA05840 (Cel1) Cryptococcus AAW41121.1 neoformans var.neoformans JEC21 xylanase II (peptide Fusarium oxysporum fragment) F3Sequence 122805 from Gibberella zeae ABT35335.1 U.S. Pat. No. 7,214,786FG03695.1 (Cel61E) Gibberella zeae PH-1 XP_383871.1 ORF (possiblefragment) Glomerella CAQ16278.1 B5WYD8 graminicola M2 ORF GlomerellaCAQ16206.1 B5WY66 graminicola M2 ORF Glomerella CAQ16208.1 B5WY68graminicola M2 ORF Glomerella CAQ16217.1 B5WY77 graminicola M2 unnamedprotein product Humicola insolens CAG27577.1 cellulase-enhancingHypocrea jecorina AAP57753.1 Q7Z9M7 factor (Cel61B) QM6A ABH82048.1ACK19226.1 ACR92640.1 endo-beta-1,4-glucanase Hypocrea jecorinaCAA71999.1 O14405.1 IV (EGIV; Egl4) RUTC-30 (Cel61A) MG05364.4Magnaporthe grisea EAA54572.1 70-15 XP_359989.1 MG07686.4 Magnaporthegrisea EAA53409.1 70-15 XP_367775.1 MG07300.4 Magnaporthe griseaEAA56945.1 70-15 XP_367375.1 MG07575.4 Magnaporthe grisea EAA53298.170-15 XP_367664.1 MG08020.4 Magnaporthe grisea EAA57051.1 70-15XP_362437.1 MG02502.4 Magnaporthe grisea EAA54517.1 70-15 XP_365800.1MG08254.4 Magnaporthe grisea EAA57285.1 70-15 XP_362794.1 MG08066.4Magnaporthe grisea EAA57097.1 70-15 XP_362483.1 MG04547.4 Magnaporthegrisea EAA50788.1 70-15 XP_362102.1 MG08409.4 Magnaporthe griseaEAA57439.1 70-15 XP_362640.1 MG09709.4 Magnaporthe grisea EAA49718.170-15 XP_364864.1 MG04057.4 Magnaporthe grisea EAA50298.1 70-15XP_361583.1 MG06069.4 Magnaporthe grisea EAA52941.1 70-15 XP_369395.1MG09439.4 Magnaporthe grisea EAA51422.1 70-15 XP_364487.1 MG06229.4Magnaporthe grisea EAA56258.1 70-15 XP_369714.1 MG07631.4 Magnaporthegrisea EAA53354.1 70-15 XP_367720.1 MG06621.4 (fragment) Magnaporthegrisea XP_370106.1 70-15 NCU07898.1 Neurospora crassa EAA33178.1 OR74AXP_328604.1 NCU05969.1 Neurospora crassa EAA29347.1 OR74A XP_325824.1NCU02916.1 Neurospora crassa EAA36362.1 OR74A XP_330104.1 NCU03000.1Neurospora crassa CAB97283.2 Q9P3R7 (B24P7.180) OR74A EAA36150.1XP_330187.1 NCU07760.1 Neurospora crassa EAA29018.1 OR74A XP_328466.1NCU07520.1 Neurospora crassa EAA29132.1 OR74A XP_327806.1 NCU01050.1Neurospora crassa CAD21296.1 Q8WZQ2 (G15G9.090) OR74A EAA32426.1XP_326543.1 NCU02240.1 Neurospora crassa EAA30263.1 OR74A XP_331016.1NCU02344.1 Neurospora crassa CAF05857.1 (B23N11.050) OR74A EAA30230.1XP_331120.1 NCU00836.1 Neurospora crassa EAA34466.1 OR74A XP_325016.1NCU08760.1 Neurospora crassa EAA26873.1 OR74A XP_330877.1 NCU07974.1Neurospora crassa EAA33408.1 OR74A XP_328680.1 NCU03328.1 Neurosporacrassa CAD70347.1 Q873G1 (B10C3.010) OR74A EAA26656.1 XP_322586.1NCU01867.1 Neurospora crassa CAE81966.1 Q7SHD9 (B13N4.070) OR74AEAA36262.1 XP_329057.1 beta-1,3-1,4-glucanase Paecilomyces (peptidefragment) thermophila J18 Pc20g11100 Penicillium CAP86439.1 B6HG02chrysogenum Wisconsin 54-1255 Pc12g13610 Penicillium CAP80988.1 B6H016chrysogenum Wisconsin 54-1255 Pc13g07400 Penicillium CAP91809.1 B6H3U0chrysogenum Wisconsin 54-1255 Pc13g13110 Penicillium CAP92380.1 B6H3A3chrysogenum Wisconsin 54-1255 Cel61 (Cel61A) Phanerochaete AAM22493.1Q8NJI9 chrysosporium BKM- F-1767 Pa_4_1020 Podospora anserina SCAP61476.1 B2ADG1 mat+ unnamed protein product Podospora anserina SCAP68173.1 B2AUV0 mat+ unnamed protein product Podospora anserina SCAP68309.1 B2AV86 mat+ unnamed protein product Podospora anserina SCAP61650.1 B2ADY5 mat+ unnamed protein product Podospora anserina SCAP68352.1 B2AVC8 mat+ Pa_7_3160 Podospora anserina S CAP68375.1 B2AVF1mat+ unnamed protein product Podospora anserina S CAP71532.1 B2B346(fragment) mat+ unnamed protein product Podospora anserina S CAP71839.1B2B403 mat+ unnamed protein product Podospora anserina S CAP72740.1B2B4L5 mat+ Pa_5_8940 Podospora anserina S CAP64619.1 B2AKU6 mat+unnamed protein product Podospora anserina S CAP73072.1 B2B5J7 mat+unnamed protein product Podospora anserina S CAP64732.1 B2AL94 mat+Pa_2_6530 Podospora anserina S CAP73254.1 B2B629 mat+ unnamed proteinproduct Podospora anserina S CAP73311.1 B2B686 mat+ unnamed proteinproduct Podospora anserina S CAP73320.1 B2B695 mat+ unnamed proteinproduct Podospora anserina S CAP64865.1 B2ALM7 mat+ unnamed proteinproduct Podospora anserina S CAP65111.1 B2AMI8 mat+ unnamed proteinproduct Podospora anserina S CAP65855.1 B2APD8 mat+ unnamed proteinproduct Podospora anserina S CAP65866.1 B2APE9 mat+ unnamed proteinproduct Podospora anserina S CAP65971.1 B2API9 mat+ unnamed proteinproduct Podospora anserina S CAP66744.1 B2ARG6 mat+ Pa_1_500 Podosporaanserina S CAP59702.1 B2A9F5 mat+ unnamed protein product Podosporaanserina S CAP61048.1 B2AC83 mat+ unnamed protein product Podosporaanserina S CAP67176.1 B2AS05 mat+ Pa_1_22040 Podospora anserina SCAP67190.1 B2AS19 mat+ unnamed protein product Podospora anserina SCAP67201.1 B2AS30 mat+ unnamed protein product Podospora anserina SCAP67466.1 B2ASU3 mat+ unnamed protein product Podospora anserina SCAP67481.1 B2ASV8 mat+ unnamed protein product Podospora anserina SCAP67493.1 B2ASX0 mat+ unnamed protein product Podospora anserina SCAP70156.1 B2AZV6 mat+ Pa_4_350 Podospora anserina S CAP61395.1 B2AD80mat+ Pa_1_16300 Podospora anserina S CAP67740.1 B2ATL7 mat+ unnamedprotein product Podospora anserina S CAP70248.1 B2AZD4 mat+ SMU2916(fragment) Sordaria macrospora k- CAQ58424.1 hell cellulase-enhancingThermoascus ABW56451.1 factor (GH61A) aurantiacus ACS05720.1 unnamedprotein product Thielavia terrestris CAG27576.1 cellulase-enhancingThielavia terrestris ACE10231.1 factor (GH61B) NRRL 8126 Sequence 4 fromU.S. Thielavia terrestris ACE10232.1 Pat. No. 7,361,495 NRRL 8126(GH61C) Sequence 4 from U.S. Thielavia terrestris ACE10232.1 Patent No.7,361,495 NRRL 8126 (GH61C) Sequence 6 from U.S. Thielavia terrestrisACE10233.1 Patent No. 7,361,495 NRRL 8126 (GH61D) Sequence 6 from U.S.Thielavia terrestris ACE10233.1 Patent No. 7,361,495 NRRL 8126 (GH61D)cellulase-enhancing Thielavia terrestris ACE10234.1 factor (131562)(GH61E) NRRL 8126 Sequence 10 from U.S. Thielavia terrestris ACE10235.1Patent No. 7,361,495 NRRL 8126 (GH61G) Sequence 10 from U.S. Thielaviaterrestris ACE10235.1 Patent No. 7,361,495 NRRL 8126 (GH61G)endoglucanase Trichoderma ADB89217.1 (EnGluIV; EndoGluIV) satumisporumEndoglucanase IV Trichoderma sp. SSL ACH92573.1 B5TYI4 endoglucanase IV(EgiV) Trichoderma viride AS ACD36973.1 B4YEW3 3.3711 endoglucanase VIITrichoderma viride AS ACD36971.1 B4YEW1 (EgvII) 3.3711 Endoglucanase II(EgII) Volvariella volvacea AAT64005.1 Q6E5B4 unknown Zea mays B73ACF78974.1 B4FA31 (ZM_BFc0036G02) ACR36748.1

GH61 proteins from Phanerochaete chrysosporium are also preferred (seeVanden Wymelenberg et al., 2009, Appl Environ Microbiol. 75(12):4058-68; Hori et al., 2011, FEMS Microbiol Lett. 321(1): 14-23).

Preferred GH61 proteins are from fungi, in particular from Thielavia,especially preferably from Thielavia terrestris or Thielaviaaurantiacus. Especially preferably said GH61 protein is GH61A fromThielavia aurantiacus or GH61B, GH61C, GH61D, GH61E or GH61G fromThielavia terrestris as described above. Other preferred GH61 proteinsinclude GH61A and B from Hypocrea jecorina (SEQ ID NOs: 15 and 16).

The GH61 protein can thus be or correspond to or comprise a naturallyoccurring GH61 protein that is found in nature or a biologically activefragment thereof. In the alternative the GH61 protein may be anon-native variant as disclosed hereinafter.

In an alternative preferred feature the oxidohydrolytic enzyme is aclass CBM33 family protein. The CBM33 family comprises acarbohydrate-binding module (CBM) which is defined as a contiguous aminoacid sequence within a carbohydrate binding protein with a discreet foldhaving carbohydrate-binding activity. For example, chitinases are knownwhich contain one or more chitin binding modules in addition tocatalytic regions. ChiA of Serratia marcescens contains a fibronectintype III—type CBM, ChiB of Serratia marcescens contains a family 5 CBMand ChiC of Serratia marcescens contains a family 12 and a fibronectintype III—like CBM. See Bourne & Henrissat, 2001, Curr. Opin. Struct.Biol. 11: 593 for domain nomenclature. Likewise, many cellulases containCBMs that bind to cellulose. Proteins binding to chitin and containingCBMs that stimulate such binding may for example be structural orsignalling molecules or they can be enzymes and the overall function ofthe protein may be determined by domains that are present in addition tothe carbohydrate binding module. The CBMs for use in methods of theinvention must, however, have oxidohydrolytic activity as defined above.So far such oxidohydrolytic activity has been detected in only one CBMfamily, namely CBM family 33. This is exemplified by the function of thechitin-binding protein (CBP) CBP21.

Members of family 33 of Carbohydrate Binding Modules (CBM33) may beidentified according to the CAZY classification system(cazy.org/CAZY/fam/acc_CBM.html), which is based on sequencesimilarities (Davies & Henrissat, 2002, Biochem Soc T 30: 291-297 andBourne & Henrissat, 2001, supra). Proteins in this family are known tobind to chitin, but binding to other polysaccharides, includingcellulose, has also been observed (Moser et al., 2008, Biotechnol.Bioeng. 100(6):1066-77). For some of these proteins it has been shownthat they act synergistically with chitinases and cellulases in thedegradation of chitin and cellulose, respectively (Vaaje-Kolstad et al.,2005, J. Biol. Chem. 280(31): 28492-7; Vaaje-Kolstad et al., 2009, FEBSJ. 276(8):2402-15; Moser et al., 2008, supra), as described in theExamples.

Studies of the action of the chitin-binding protein CBP21 (and otherCBM33 proteins) have now led to the identification of CBM33 proteins asoxidohydrolases.

As described herein, all members of CBM family 33 contain a family 33carbohydrate binding module. In several cases, the CBM33 module makes upthe whole protein, i.e., the protein consists of or consists essentiallyof a single family 33 CBM, which is in nature synthesized and secretedas such. However some family 33 CBMs may be fused to one or moreadditional non-catalytic carbohydrate binding modules (e.g., CBM family2, CBM family 3 and CBM family 5 modules). These proteins are bi- ormulti-domain proteins. There is also one known example of a family 33carbohydrate binding module that is present as an individual modulewithin a much larger catalytic protein. This is the beta-1,4-mannanaseprotein of Caldibacillus cellulovorans (Sunna et al., 2000, Appl.Environ. Micro. 66(2): 664-670).

The family 33 CBMs are usually approximately 150-250 amino acids, e.g.,160-240, 170-230, 180-220, 190-210 amino acids in size and have amolecular weight of approximately 20 kDa, preferably 19-21 kDa, 18-21kDa, 19-22 kDa or 18-20 kDa in size, though CBM33 domains as large as300-400 amino acids with a molecular weight of approximately 30-40 kDamay also be used. The size of a protein can readily be determined bystandard methods that are known in the art.

Preferably, the oxidohydrolytic enzyme consists of a single family 33CBM, or consists essentially of a family 33 CBM.

If said oxidohydrolytic enzyme “consists essentially of” a family 33CBM, it is meant that additional amino acids may be present in theprotein, in addition to those that make up the family 33 CBM. Preferablythere are 1-3, 1-5, 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70,70-80, 80-90 or 90-100 or more additional amino acids present. Theseadditional amino acids are in general present C terminal to the family33 CBM.

Alternatively, the oxidohydrolytic enzyme can comprise a family 33 CBM.Additional modules or domains may thus be present in the protein.Examples of such modules are CBM family 2, CBM family 3 and CBM family 5modules. If additional domains or modules are present, they are ingeneral found C-terminal to the family 33 CBM.

Thus in a preferred aspect, the oxidohydrolytic enzyme can contain,consist or consist essentially of a naturally occurring family 33 CBM(or CBM33 family protein) such as CBP21 (or to a homologue thereof fromanother species) or a biologically active fragment thereof. It canalternatively contain, consist or consist essentially of a variant of anaturally occurring family 33 CBM (or CBM33 family protein) or abiologically active fragment thereof.

Oxidohydrolytic enzymes which comprise or consist of a family 33 CBMmodule or the full family 33 CBM protein (which comprises the family 33CBM module) or its fragments or variants are referred to herein,collectively, as CBM33 proteins or CBM33 family members or proteins.

Naturally occurring CBM33 proteins that can be used in the inventioninclude microbial (e.g., bacterial), eukaryotic (e.g., Dictyostelium) orviral CBM33 proteins. Bacterial CBM33 proteins are, however, preferred.

Examples of known CBM33 proteins which may be used in methods of theinvention and relevant database accession numbers (which are herebyincorporated by reference) are set out in Table 1:

PROTEIN ORGANISM GENBANK/GENPEPT BACTERIA Cbp1 Alteromonas sp. O-7AB063629 BAB79619.1 chitin binding protein ChbA Bacillusamyloliquefaciens ALKO AF181997 AAG09957.1 2718 BA_3348 Bacillusanthracis str. A2012 NC_003995 NP_656708.1 BA2827 Bacillus anthracisstr. Ames AE017033 AAP26659.1 NC_003997 NP_845173.1 BA2793 Bacillusanthracis str. Ames AE017032 AAP26628.1 NC_003997 NP_845142.1 GBAA2827Bacillus anthracis str. Ames 0581 AE017334 AAT31944.1 GBAA2793 Bacillusanthracis str. Ames 0581 AE017334 AAT31910.1 BAS2636 Bacillus anthracisstr. Sterne AE017225 AAT54946.1 BAS2604 Bacillus anthracis str. SterneAE017225 AAT54914.1 BCE2855 Bacillus cereus ATCC 10987 AE017273AAS41767.1 NC_003909 NP_979159.1 BCE2824 Bacillus cereus ATCC 10987AE017273 AAS41736.1 NC_003909 NP_979128.1 BC2827 Bacillus cereus ATCC14579 AE017007 AAP09778.1 NC_004722 NP_832577.1 BC2798 Bacillus cereusATCC 14579 AE017007 AAP09751.1 NC_004722 NP_832550.1 pE33L466_0276(ChbA) Bacillus cereus E33L CP000040 AAY60428.1 BTZK2523 (ChB) Bacilluscereus ZK CP000001 AAU17736.1 BTZK2552 (ChB) Bacillus cereus ZK CP000001AAU17707.1 ABC1161 Bacillus clausii KSM-K16 AP006627 BAD63699.1 BH1303Bacillus halodurans C-125 AP001511 BAB05022.1 NC_002570 NP_242169.1BLi00521 or BL00145 Bacillus licheniformis DSM 13 ATCC CP000002AAU22121.1 14580 AE017333 AAU39477.1 BT9727_2586 (ChB) Bacillusthuringiensis serovar AE017355 AAT61310. 1 konkukian str. 97-27BT9727_2556 (ChB) Bacillus thuringiensis serovar AE017355 AAT61323.1konkukian str. 97-27 BMAA1785 Burkholderia mallei ATCC 23344 CP000011AAU45854.1 BMA2896 Burkholderia mallei ATCC 23344 CP000010 AAU48386.1BURPS1710b_0114 Burkholderia pseudomallei 1710b CP000124 ABA49030.1BURPS1710b_A2047 Burkholderia pseudomallei 1710b CP000125 ABA53645.1BPSL3340 Burkholderia pseudomallei K96243 BX571965 CAH37353.1 BPSS0493Burkholderia pseudomallei K96243 BX571966 CAH37950.1 Bcep18194_C6726Burkholderia sp. 383 CP000150 ABB05775.1 BTH_II1925 Burkholderiathailandensis E264; CP000085 ABC34637.1 ATCC 700388 BTH_I3219Burkholderia thailandensis E264; CP000086 ABC38514.1 ATCC 700388Beta-1,4-mannanase Caldibacillus cellulovorans AF163837 AAF22274.1(ManA) CV0554 Chromobacterium violaceum ATCC AE016911 AAQ58230.1 12472NC_005085 NP_900224.1 CV0553 Chromobacterium violaceum ATCC AE016911AAQ58229.1 12472 NC_005085 NP_900223.1 CV2592 (CpbD) Chromobacteriumviolaceum ATCC AE016919 AAQ60262.1 12472 NC_005085 NP_902262.1 CV3489Chromobacterium violaceum ATCC AE016922 AAQ61150.1 12472 NC_005085NP_903159.1 CV3323 (CbpD1) Chromobacterium violaceum ATCC AE016921AAQ60987.1 12472 NC_005085 NP_902993.1 EF0362 Enterococcus faecalis V583AE016948 AA080225.1 NC_004668 NP_814154.1 Sequence 4287 from U.S.Enterococcus faecium — AAQ43729.1 Pat. No. 6,583,275 FTL_1408Francisella tularensis subsp. AM233362 CAJ79847.1 holarctica LVSFTT0816c Francisella tularensis subsp. AJ749949 CAG45449.1 tularensisSchu 4 HCH_00807 Hahella chejuensis KCTC 2396 CP000155 ABC27701.1HCH_03973 Hahella chejuensis KCTC 2396 CP000155 ABC30692.1 Ip_1697Lactobacillus plantarum WCFS1 AL935256 CAD64126.1 NC_004567 NP_785278.1LSA1008 Lactobacillus sakei subsp. sakei 23K CR936503 CAI55310.1 YucGLactococcus lactis subsp. lactis AE006425 AAK06049.1 IL1403 NC_002662NP_268108.1 Ipp0257 Legionella pneumophila Paris CR628336 CAH11404.1Lin2611 Listeria innocua AL596173 CAC97838.1 NC_003212 NP_471941.1Lmo2467 Listeria monocytogenes EGD-e AL591983 CAD00545.1 NC_003210NP_465990.1 LMOf2365_2440 Listeria monocytogenes str. 4b AE017330AAT05205.1 F2365 OB0810 Oceanobacillus iheyensis HTE831 AP004595BAC12766.1 NC_004193 NP_691731.1 PBPRB0312 Photobacterium profundum SS9CR378676 CAG22185.1 plu2352 Photorhabdus luminescens subsp. BX571866CAE14645.1 laumondii TTO1 NC_005126 NP_929598.1 Sequence 6555 from U.S.Proteus mirabilis — AAR43285.1 Pat. No. 6,605,709 chitin-binding proteinChiB Pseudoalteromonas sp. S9 AF007895 AAC79666.1 chitin-binding proteinPseudomonas aeruginosa PAO1 AE004520 AAG04241.1 (CbpD; PA0852) NC_002516NP_249543.1 chitin-binding protein Pseudomonas aeruginosa PAO25 AF196565AAF12807.1 (CbpD) PFL_2090 Pseudomonas fluorescens Pf-5 CP000076AAY91365.1 Pfl_3569 Pseudomonas fluorescens PfO-1 CP000094 ABA75307.1Psyr_2856 Pseudomonas syringae pv. syringae CP000075 AAY37892.1 B728aPSPTO2978 Pseudomonas syringae pv. tomato AE016866 AAO56470.1 str.DC3000 NC_004578 NP_792775.1 RF_0708 Rickettsia felis URRWXCal2 CP000053AAY61559.1 chitin-binding protein Saccharophagus degradans 2-40 BK001045DAA01337.1 (CbpA) ORF Salinivibrio costicola 5SM-1 AY207003 AAP42509.1chitin-binding protein Serratia marcescens 2170 AB015998 BAA31569.1(Cbp21) chitin-binding protein Serratia marcescens BJL200 AY665558AAU88202.1 (Cbp21) ORF2 Serratia marcescens KCTC2172 L38484 AAC37123.1SO1072 Shewanella oneidensis MR-1 AE015551 AAN54144.1 NC_004347NP_716699.1 SG1515 (possible Sodalis glossinidius str. ‘morsitans’AP008232 BAE74790.1 fragment) SAV6560 Streptomyces avermitilis MA-4680AP005047 BAC74271.1 NC_003155 NP_827736.1 SAV2168 Streptomycesavermitilis MA-4680 AP005029 BAC69879.1 NC_003155 NP_823344.1 SAV5223(Chb) Streptomyces avermitilis MA-4680 AP005042 BAC72935.1 NC_003155NP_826400.1 SAV2254 (CelS2) Streptomyces avermitilis MA-4680 AP005030BAC69965.1 NC_003155 NP_823430.1 SCO7225 or SC2H12.24 Streptomycescoelicolor A3(2) AL359215 CAB94648.1 NC_003888 NP_631281.1 SCO6345 orSC3A7.13 Streptomyces coelicolor A3(2) AL031155 CAA20076.1 NC_003888NP_630437.1 SCO2833 (Chb) Streptomyces coelicolor A3(2) AL136058CAB65563.1 NC_003888 NP_627062.1 SCO0643 or SCF91.03c Streptomycescoelicolor A3(2) AL132973 CAB61160.1 NC_003888 NP_624952.1 SCO0481 orSCF80.02 Streptomyces coelicolor A3(2) AB017013 BAA75647.1 AL121719CAB57190.1 NC_003888 NP_624799.1 SCO1734 or SCI11.23 Streptomycescoelicolor A3(2) AL096849 CAB50949.1 NC_003888 NP_626007.1 CelS2(SCO1188 or Streptomyces coelicolor A3(2) AL133210 CAB61600.1 SCG11A.19)NC_003888 NP_625478.1 chitin binding protein Streptomyces griseusAB023785 BAA86267.1 cellulose binding protein Streptomyces halstediiU51222 AAC45430.1 (ORF2) chitin-binding protein Streptomycesolivaceoviridis ATCC X78535 CAA55284.1 11238 chitin binding proteinStreptomyces reticuli Y14315 CAA74695.1 (Chb2) chitin-binding proteinStreptomyces thermoviolaceus OPC- AB110078 BAD01591.1 (Cbp1) 520chitin-binding protein celS2 Streptomyces viridosporus AF126376AAD27623.1 Tfu_1665 (E8) Thermobifida fusca YX CP000088 AAZ55700.1Tfu_1268 (E7) Thermobifida fusca YX CP000088 AAZ55306.1 VCA0140 Vibriocholerae N16961 AE004355 AAF96053.1 NC_002506 NP_232540.1 VCA0811 Vibriocholerae N16961 AE004409 AAF96709.1 NC_002506 NP_233197.1 VFA0143 Vibriofischeri ES114 CP000021 AAW87213.1 VFA0013 Vibrio fischeri ES114CP000021 AAW87083.1 VPA0092 Vibrio parahaemolyticus RIMDA P005084BAC61435.1 2210633 NC_004605 NP_799602.1 VPA1598 Vibrio parahaemolyticusRIMDA P005089 BAC62941.1 2210633 NC_004605 NP_801108.1 VV21258 Vibriovulnificus CMCP6 AE016812 AAO08152.1 NC_004460 NP_763162.1 VV20044Vibrio vulnificus CMCP6 AE016808 AAO07021.1 NC_004460 NP_762031.1VVA0086 Vibrio vulnificus YJ016 AP005344 BAC96112.1 NC_005140NP_936142.1 VVA0551 Vibrio vulnificus YJ016 AP005346 BAC96577.1NC_005140 NP_936607.1 ChiY Yersinia enterocolitica (type 0:8) WA-AJ344214 CAC83040.2 314 YP0706 Yersinia pestis biovar Medievalis str.AE017129 AAS60972.1 91001 NC_005810 NP_992095.1 YPO3227 Yersinia pestisCO92 AJ414156 CAC92462.1 NC_003143 NP_406699.1 Y0962 Yersinia pestis KIMAE013699 AAM84543.1 NC_004088 NP_668292.1 YPTB3366 Yersiniapseudotuberculosis IP BX936398 CAH22604.1 32953 YPTB0899 Yersiniapseudotuberculosis IP BX936398 CAH20139.1 32953 EUKARYOTA ORF-26 Agrotissegetum DQ123841 AAZ38192.1 nucleopolyhedrovirus spheroidin-like proteinAutographa californica L22858 AAA66694.1 (Gp 37) nucleopolyhedrovirusD00583 BAA00461.1 NC_001623 NP_054094.1 Fusolin Bombyx mori nuclearpolyhedrosis U55071 AAB47606.1 virus L33180 AAC63737.1 NC_001962NP_047468.1 Spheroidin Choristoneura biennis M34140 AAA42887.1entomopoxvirus VIRUSES ORF-26 Agrotis segetum DQ123841 AAZ38192.1nucleopolyhedrovirus Spheroidin-like protein Autographa californicaD00583 BAA00461.1 (Gp 37) nucleopolyhedrovirus L22858 AAA66694.1NC_001623 NP_054094.1 Fusolin Bombyx mori nuclear polyhedrosis U55071AAB47606.1 virus NC_001962 NP_047468.1 L33180 AA063737.1 SpheroidinChoristoneur biennis M341140 AAA42887.1 entomopoxvirus ORF60Choristoneura fumiferana defective AY327402 AAQ91667.1nucleopolyhedrovirus NC_005137 NP_932669.1 spindle-like proteinChoristoneura fumiferana nuclear U26734 AA055636.1 polyhedrosis virusNC_004778 NP_848371.1 GP37 (ORF-67 GP37) Chrysodeixis chalcites AY864330AAY83998.1 nucleopolyhedrovirus ORF57 Epiphyas postvittana AY043265AAK85621.1 nucleopolyhedrovirus NC_003083 NP_203226.1 GP37 Helicoverpaarmigera single AF266696 AAK57880.1 nucleocapsid polyhedrovirus AF303045AAK96305.1 NC_003094 NP_203613.1 ORF59 Helicoverpa zea AF334030AAL56204.1 nucleopolyhedrovirus NC_003349 NP_542682.1 gp37 Heliocoverpaarmigera AF271059 AAG53801.1 nucleopolyhedrovirus G4 NC_002654NP_075127.1 Fusolin Heliothis armigera entomopoxvirus L08077 AAA92858.1HynVgp086 (slp) Hyphantria cunea AP009046 BAE72375.1nucleopolyhedrovirus Gp37 protein Leucania separata nuclear AB009614BAA24259.1 polyhedrosis virus fusolin-like protein Lymantria disparU38895 AAB07702.1 nucleopolyhedrovirus AF081810 AA070254.1 NC_001973NP_047705.1 gp37 protein Mamestra brassicae AF108960 AAD45231.1nucleopolyhedrovirus ORF 37 (Gp37) Mamestra configurata U59461AAM09145.1 nucleopolyhedrovirus A AF539999 AAQ11056.1 Gp37 Mamestraconfigurata AY126275 AAM95019.1 nucleopolyhedrovirus B NC_004117NP_689207.1 spheroidin-like protein Orgyia pseudotsugata nuclear U75930AA059068.1 (Gp 37) polyhedrosis virus D13306 BAA02566.1 NC_001875NP_046225.1 enhancing factor Pseudaletia separata D50590 BAA09138.1entomopoxvirus ORF25 Spodoptera exigua AF169823 AAF33555.1nucleopolyhedrovirus NC_002169 NP_037785.1 gp37 (fragment) Spodopterafrugiperda MNPV AY250076 AAP79107.1 ubiquitin GP37 fusion Spodopteralitura AF325155 AAL01718.1 protein nucleopolyhedrovirus G2 NC_003102NP_258300.1 gp37 Trichoplusia ni single DQ017380 AAZ67435.1nucleopolyhedrovirus Fusolin unidentified entomopoxvirus X77616CAA54706.1 ORF107 Xestia c-nigrum granulovirus AF162221 AAF05221.1NC_002331 NP_059255.1 Other preferred bacterial CBM33 proteins include:Cfla_0175 Cellulomonas flavigena DSM ADG73094.1 D5UGB1 20109 Cfla_0172Cellulomonas flavigena DSM ADG73091.1 D5UGA8 20109 Cfla_0316Cellulomonas flavigena DSM ADG73234.1 D5UH31 20109 Cfla_0490Cellulomonas flavigena DSM ADG73405.1 D5UHY1 20109 CJA_2191 (Cbp33A)Cellvibrio japonicus Ueda107 ACE83992.1 B3PJ79 CJA_3139 (cbp33/10B)Cellvibrio japonicus Ueda107 ACE84760.1 B3PDT6

Bacterial CBM33 proteins can be from any appropriate source but arepreferably from a genus selected from the group consisting of Bacillus,Chromobacterium, Enterococcus, Francisella, Hahella, Lactobacillus,Lactococcus, Legionella, Listeria, Oceanobacillus, Photobacterium,Photothabdus, Proteus, Pseudoalteromonas, Pseudomonas, Rickettsia,Saccharophagus, Salinvibrio, Serratia, Shewanella, Sodalis,Streptomyces, Thermobifida, Vibrio and Yersini and optionallyCellulomonas and Cellvibrio.

Preferably said CBM33 protein is a CBP21 as described in U.S. PatentApplication No. 2007/0218046 which is incorporated herein by reference.For example the CBP21 of Serratia marescens (SEQ ID NO: 4) is preferred.Alternatively, the EfCBM33 of Enterococcus faecalis (SEQ ID NO: 5), E7of Thermobifida fusca (SEQ ID NO: 6), CelS2 of Streptomyces coelicolorA3(2) (SEQ ID NO: 7), Cfla_0175 of Cellulomonas flavigena DSM 20109)(SEQ ID NO: 8), Cfla_0172 of Cellulomonas flavigena DSM 20109) (SEQ IDNO: 9), Cfla_0316 of Cellulomonas flavigena DSM 20109) (SEQ ID NO: 10),Cfla_0490 of Cellulomonas flavigena DSM 20109) (SEQ ID NO: 11), CJA_2191(Cbp33A) of Cellvibrio japonicus Ueda107 (SEQ ID NO: 12), CJA_3139(Cbp33/10B) of Cellvibrio japonicus Ueda107 (SEQ ID NO: 13) and SC01734of Streptomyces coelicolar A3(2)) (SEQ ID NO: 14), may be used. ChbA ofB. amyloliquefaciens (Chu et al., 2001, Microbiology 147 (Pt7):1793-803) CHB1, 2 & 3 of Streptomyces (Svergun et al., 2000,Biochemistry 39(35):10677-83, Zeltins et al., 1997, Eur. J. Biochem.246(2):557-64, Zeltins et al., 1995, Anal. Biochem. 231(2):287-94,Schnellmann et al., 1994, Mol. Microbiol. 13(5):807-19; Kolbe et al.,1998, Microbiology 144 (Pt 5):1291-7; Saito et al., 2001, Appl. Environ.Microbiol. 67(3):1268-73) and CBP1 of Alteramonas (Tsujibo et al., 2002,Appl. Environ. Microbiol. 68:263-270) are also preferred CBM33 proteinsfor use in the invention. All of these references are incorporatedherein by reference.

The oxidohydrolytic enzyme can thus be or correspond to or comprise anaturally occurring CBM33 family protein (such as CBP21, EfCBM33, ChbA,CHB1, 2 & 3 and CBP1 or E7, CelS2, Cfla_0175, Cfla_0172, Cfla_0316,Cfla_0490, CJA_2191 (Cbp33A), CJA_3139 (Cbp33/10B) and SCO1734) or GH61family protein in that it is found in nature or a biologically activefragment thereof. In the alternative the oxidohydrolytic enzyme may be anon-native variant as disclosed hereinafter.

As mentioned above, the oxidohydrolytic enzymes may be native proteinsor biologically active fragments thereof or molecules containing thoseenzymes. Furthermore, non-native proteins may be derived from anaturally occurring protein, e.g., from a GH61 or CBM33 family protein.

Such fragments are preferably at least 200, 300 or 400 amino acids inlength and preferably comprise simple, short deletions from the N of Cterminal, e.g., a C terminal deletion of 1, 2, 3, 4 or 5 amino acids.

All such variants or fragments must retain the functional property ofthe protein from which they are derived such that they are “biologicallyactive”. Thus they must retain oxidohydrolytic activity, e.g., under theconditions described in the Examples (e.g., exhibit enhanced activitywhen used in the presence of a reducing agent and one or moresaccharolytic enzymes when compared to performing the method without thereducing agent, see, e.g., FIG. 4). Furthermore, said biologicallyactive fragments and variants must be able to enhance degradation asdescribed herein, i.e., enhance degradation of the polysaccharidesubstrate (e.g., when said degradation is performed in the presence ofone or more saccharolytic enzymes) when used in the presence of areducing agent and a divalent metal ion, relative to degradationomitting the reducing agent and divalent metal ion. Some loss ofactivity is contemplated, e.g., the biologically active fragment orvariant may have at least 50, 60, 70, 80, 90 or 95% of theoxidohydrolytic activity of the native full length sequence wherein saidactivity may be assessed in terms of the extent or level of degradation,e.g., hydrolysis, achieved over a set time period, e.g., as assessed bythe production of reaction products such as oligo and/or di-saccharides.

Variants include or comprise naturally occurring variants of theoxidohydrolytic enzymes described above such as comparable proteins orhomologues found in other species or more particularly variants foundwithin other microorganisms, which have the functional properties of theenzymes as described above.

Variants of the naturally occurring oxidohydrolytic enzymes as definedherein can also be generated synthetically, e.g., by using standardmolecular biology techniques that are known in the art, for examplestandard mutagenesis techniques such as site directed or randommutagenesis. Such variants further include or comprise proteins havingat least 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequencesimilarity or identity with a naturally occurring oxidohydrolytic enzymeat the amino acid level.

Thus in a preferred aspect the oxidohydrolytic enzyme for use in themethods described herein is a polypeptide which comprises an amino acidsequence as set forth in any one of SEQ ID NOs: 1 to 14 (e.g., SEQ IDNOs: 1-4 or 1-5) and/or 15 to 16 (optionally with or without the leaderpeptide, where present) or a sequence with at least 30, 40, 50, 60, 70,80, 90, 95, 97, 98 or 99% sequence identity thereto or a biologicallyactive fragment thereof comprising at least 100 amino acids (preferablyat least 200 or 300 amino acids) of said sequence.

In the sequences below, the leader peptides, where present, areunderlined.

GH61E from T. terrestris (Acc. No. ACE10234) SEQ ID NO: 1MLANGAIVFLAAALGVSG HYTWPRVNDGADWQQVRKADNWQDNGYVGDVTSPQIRCFQATPSPAPSVLNTTAGSTVTYWANPDVYHPGPVQFYMARVPDGEDINSWNGDGAVWFKVYEDHPTFGAQLTWPSTGKSSFAVPIPPCIKSGYYLLRAEQIGLHVAQSVGGAQFYISCAQLSVTGGGSTEPPNKVAFPGAYSATDPGILINIYYPVPTSYQNPGPAVFSCMLANGAIVFLAAALGVSGHYTWPRVNDGADWQQVRKADNWQDNGYVGDVTSPQIRCFQATPSPAPSVLNTTAGSTVTYWANPDVYHPGPVQFYMARVPDGEDINSWNGDGAVWFKVYEDHPTFGAQLTWPSTGKSSFAVPIPPCIKSGYYLLRAEQIGLHVAQSVGGAQFYISCAQLSVTGGGSTEPPNKVAFPGAYSATDPGILINIYYPVPTSYQNPGPAVF SCGH61A from T. aurantiacus (Acc. No. ABW56451) SEQ ID NO: 2MSFSKIIATAGVLASASLVAG HGFVQNIVIDGKKYYGGYLVNQYPYMSNPPEVIAWSTTATDLGFVDGTGYQTPDIICHRGAKPGALTAPVSPGGTVELQWTPWPDSHHGPVINYLAPCNGDCSTVDKTQLEFFKIAESGLINDDNPPGIWASDNLIAANNSWTVTIPTTIAPGNYVLRHEIIALHSAQNQDGAQNYPQCINLQVTGGGSDNPAGTLGTALYHDTDPGILINIYQKLSSYIIPGPPLYTGGH61B from T. terrestris (Acc. No. ACE10231) SEQ ID NO: 3MKSFTIAALAALWAQEAAA HATFQDLWIDGVDYGSQCVRLPASNSPVTNVASDDIRCNVGTSRPTVKCPVKAGSTVTIEMHQQPGDRSCANEAIGGDHYGPVMVYMSKVDDAVTADGSSGWFKVFQDSWAKNPSGSTGDDDYWGTKDLNSCCGKMNVKIPEDIEPGDYLLRAEVIALHVAASSGGAQFYMSCYQLTVTGSGSATPSTVNFPGAYSASDPGILINIHAPMSTYVVPGPTVYAGGSTKSAGSSCSGCEATCTVGSGPSATLTQPTSTATATSAPGGGGSGCTAAKYQQCGGTGYTGCTTCASGSTCSAVSPPYYSQCL CBP21 of Serratia marescens SEQ ID NO: 4MNKTSRTLLSLGLLSAAMFGVSQQANA HGYVESPASRAYQCKLQLNTQCGSVQYEPQSVEGLKGFPQAGPADGHIASADKSTFFELDQQTPTRWNKLNLKTGPNSFTWKLTARHSTTSWRYFITKPNWDASQPLTRASFDLTPFCQFNDGGAIPAAQVTHQCNIPADRSGSHVILAVWDIADTANAFYQAIDVNLSK

In the above sequence (SEQ ID NO: 4), amino acid residues 1 to 27correspond to the leader peptide that is necessary for secretion of theprotein in a natural system and amino acids 28-196 correspond to themature protein. Using Pfam for domain/module discovery (“The Pfamprotein families database” by Finn et al., 2010, Nucleic Acids ResearchDatabase Issue 38: D211-222), for SEQ ID NO: 4 residues 28-194, i.e.,essentially the complete mature protein, are classified as CBM33.Similarly, in relation to the sequence presented in SEQ ID NO: 5, below,the mature protein starts at position 29 (H).

EfCBM33 from Enterococcus faecalis (Acc. No. Q838S1) SEQ ID NO: 5MKKSLLTIVLAFSFVLGGAALAPTVSEA HGYVASPGSRAFFGSSAGGNLNTNVGRAQWEPQSIEAPKNTFITGKLASAGVSGFEPLDEQTATRWHKTNITTGPLDITWNLTAQHRTASWDYYITKNGWNPNQPLDIKNFDKIASIDGKQEVPNKVVKQTINIPTDRKGYHVIYAVWGIGDTVNAFYQAIDVNIQ E7 from Thermobifida fusca (Acc. No. Q47QG3) SEQ ID NO: 6MHRYSRTGKHRWTVRALAVLFTALLGLTQWTAPASA HGSVINPATRNYGCWLRWGHDHLNPNMQYEDPMCWQAWQDNPNAMWNWNGLYRDWVGGNHRAALPDGQLCSGGLTEGGRYRSMDAVGPWKTTDVNNTFTIHLYDQASHGADYFLVYVTKQGFDPTTQPLTWDSLELVHQTGSYPPAQNIQFTVHAPNRSGRHVVFTIWKASHMDQTYYLCSDVNFV CelS2 from Streptomyces coeficolor A3(2)(Acc. No. Q9RJY2) SEQ ID NO: 7 MVRRTRLLTLAAVLATLLGSLGVTLLLGQGRAEAHGVAMMPGSRTYLCQL DAKTGTGALDPTNPACQAALDQSGATALYNWFAVLDSNAGGRGAGYVPDGTLCSAGDRSPYDFSAYNAARSDWPRTHLTSGATIPVEYSNWAAHPGDFRVYLTKPGWSPTSELGWDDLELIQTVTNPPQQGSPGTDGGHYYWDLALPSGRSGDALIFMQWVRSDSQENFFSCSDVVFDGGNGEVTGIRGSGSTPDPDPTPTPTDPTTPPTHTGSCMAVYSVENSWSGGFQGSVEVMNHGTEPLNGWAVQWQPGGGTTLGGVWNGSLTSGSDGTVTVRNVDHNRVVPPDGSVTFGFTATST GNDFPVDSIGCVAP

The signal peptide for the proteins in SEQ ID NOs: 1, 2, 3, 4, 5, 6 and7 is underlined. The two conserved histidines in the metal binding motifof these proteins are shown in bold formatting. The signal peptides andhistidines are similarly shown for SEQ ID Nos: 8-14, below.

Cfla_0175 from Cellulomonas flavigena DSM 20109 SEQ ID NO: 8MPRHRSTRRALAGLAATAVVTTALVTVPTVAQA HGGLTNPPTRTYACYQDGLAGGAAAGEAGNIRPRNAACVNAFDNEGNYSFYNWYGNLLGTIAGRHETIADGKVCGPDARFASYNTPSSAWPTTKVTPGQTMTFQYAAVARHPGWFTTWITKDGWNQNEPIGWDDLEPAPFDRVLDPPLREGGPAGPEYWWNVKLPSNKSGKHVLFNIWERTDSPESFYNCVDVDFGGGGTVTPSPTPSVTPTRTPTPSPTPSVTPSPTPSVTPTPTPTPTPTPSPTPTLTVTPTPTPTSVPGDSVCELEVDTSSAWPGGFQGTVTVFNATMEPVNGWQVSWKFTNGETIAQSWSGVTSQSGSTVTVKNADWNSTIAHHNAVNFGFIGSGTPKAVTDATLNGKPCIVRCfla_0172 from Cellulomonas flavigena DSM 20109 SEQ ID NO: 9MFIPTRSRFGRLARLALAVPLALAATGIVATSASA HGSVTDPPSRNYGCWEREGGTHMDPAMAQRDPMCWQAFQANPNTMWNWNGNFREGVGGRHEQVIPDDQLCSAGKTQNGLYASLDTPGPWIMKTVPHNFTLTLTDGAMHGADYMRIYVSKAGYDPTTDPLGWDDIELIKETGRYGTTGLYQADVSIPSNRTGRAVLFTIWQASHLDQPYYICSDININGTAPTQQPTQQPTQQPTQQPTQQPTQQPTQQPTQQPTQQPTQQPTQNPGTGACTATVKAASTWGNGWQGEVTVTAGSSAINGWKVTVGGASITQAWSGSYSGGTFSNAEWNGKLAAGASTTAGFIASG TPGTLTATCTAACfla_0316 from Cellulomonas flavigena DSM 20109 SEQ ID NO: 10MSRISPLRRVAAACGALAIGAATVVGSIALAAPASA HGAVSDPPSRIYGCWERWASNFTDPAMATSDPQCWDAWQSEPQAMWNWNGMFKEGAAGQHEQSIPDGKLCSADNPLYAAADDPGPWRTTPVDHDFRLTLHDPSNHGADYLKIYVTKQGYDARSEALTWADLELVKTTGRYATSSPYVTDVSVPRDRTGHHVVFTIWQASHLDQPYYQCSDVTFGGGGTPTTSPTTPAPTPTTPAPTTPAPTPTTPAPTTPAPTTPAPTTPAPTTPAPTQPADGACTAAIEVVSAWQGGYQATVTVTAGSGGLDGWTVTVPGATITQAWNGTATGSTITAAGWNGTVAAGGTAGV GFLGSGSPDGLTATCAAACfla_0490 from Cellulomonas flavigena DSM 20109 SEQ ID NO: 11MRSHALPRSARPTPGRLLLSVLAVIALAFAVLTVAPAPSAQA HGWISDPPSRQDLCYTGAVSNCGPVMYEPWSVEAKKGSMQCSGGGRFTELDNESRSWPRQNLKTNQVFTWDIVANHSTSTWEYFVDGRLHTTIDDKGALPPNRFTHTINNLPEGNHKIFVRWNIADTVNAFYQCIDAYITPGGTPGPTQQPTQQPTQQPTQQPTQQPTQQPTQQPTQQPGNGACTATFKTNNAWGNGYQGEITVTAGSSAIRGWKVTVNGATITQAWSSQLSGSTLSNASWNGSLNAGASTTLGFIAN GTPSGVTATCAAACJA_2191 (Cbp33A) from Cellvibrio japonicus Ueda107 SEQ ID NO: 12MFNTRHLLAGVSQLVKPASMMILAMASTLAIHEASA HGYVSSPKSRVIQCKENGIENPTHPACIAAKAAGNGGLYTPQEVAVGGVRDNHDYYIPDGRLCSANRANLFGMDLARNDWPATSVTPGAREFVWTNTAAHKTKYFRYYITPQGYDHSQPLRWSDLQLIHDSGPADQEWVSTHNVILPYRTGRHIIYSIWQRDWDRDAAEGFYQCIDVDFGNGTGTGSSSSVASSVVSSVTSSSVASSVASSLSNDTCATLPSWDASTVYTNPQQVKHNSKRYQANYWTQNQNPSTNSGQYGPWLDLGNCVTSGGSSSVASSSVASSVASSVTSSVASSVVSGNCISPVYVDGSSYANNALVQNNGSEYRCLVGGWCTVGGPYAPGTGWAWANAWELVRSCQ CJA_3139 (Cbp33/10B) from Cellvibrio japonicus Ueda107 SEQ ID NO: 13MNNKFVKMGGMGALLLAFSALSFG HGFVDSPGARNYFCGAVTKPDHVMNGVARYPECAGAFANDFNGGYSYMSVLTHHQGRKVLGPVARNVCGFDSETWNGGKTPWDNAINWPVNNINSGTLTFSWDISNGPHFDDTSDFRYWITKPGFVYQVGRELTWADFEDQPFCDLAYNDDNPGAYPNVRADKPNTHFHTTCTVPARTGRHVIYAEWGREPPTYERFHGCIDVQIGGGSNSSVPVSSSSSSRSSSSSSLAPSSSSRSSSSSSSVSSSRSSSSSVVSSSSSSRPASSSSSSTGGSTEYCNVVYGWQVAICKNTTSGWSNENQQTCIGRDTCNAPRSCO1734 from Streptomyces coelicolor A3(2) SEQ ID NO: 14MPAPSASRRAAAVAVAGLAPLALTTLAAAPASA HGSMGDPVSRVSQCHAEGPENPKSAACRAAVAAGGTQALYDWNGIRIGNAAGKHQELIPDGRLCSANDPAFKGLDLARADWPATGVSSGSYTFKYRVTAPHKGTFKVYLTKPGYDPSKPLGWGDLDLSAPVATSTDPVASGGFYTFSGTLPERSGKHLLYAVWQRSDSPEAFYSCSDVTFGGDGDGDGDGGSGSGAATGDDTASGDAEAGAAPAPEASAPSEEQLAAAAEKSTIEHHGHGDQDAATTTDPTDPAAAPEEAPGTAAEPHQVKAAGGGTENLAETGGDSTTPYIAVGGAAALALGAAVLFASVRRRATT GGRHGHSEQ ID NOs: 15 and 16 are shown without a leaderpeptide. The two conserved histidines in the metalbinding motif of these proteins are shown in bold formatting.HjGH61A from Hypocrea jecorina SEQ ID NO: 15HGHINDIVINGVWYQAYDPTTFPYESNPPIVVGWTAADLDNGFVSPDAYQNPDIICHKNATNAKGHASVKAGDTILFQWVPVPWPHPGPIVDYLANCNGDCETVDKTTLEFFKIDGVGLLSGGDPGTWASDVLISNNNTWVVKIPDNLAPGNYVLRHEIIALHSAGQANGAQNYPQCFNIAVSGSGSLQPSGVLGTDLYHATDPGVLINIYTSPLNYIIPGPTVVSGLPTSVAQGSSAATATASATVPGGGSGPTSRTTTTARTTQASSRPSSTPPATTSAPAGGPTQTLYGQCGGSGYSGPTRCAPPATCSTLNPYYAQCLN HjGH61 from Hypocrea jecorina SEQ ID NO: 16HGQVQNFTINGQYNQGFILDYYYQKQNTGHFPNVAGWYAEDLDLGFISPDQYTTPDIVCHKNAAPGAISATAAAGSNIVFQWGPGVWPHPYGPIVTYVVECSGSCTTVNKNNLRVVVKIQEAGINYNTQVWAQQDLINQGNKWTVKIPSSLRPGNYVFRHELLAAHGASSANGMQNYPQCVNIAVTGSGTKALPAGTPATQLYKPTDPGILFNPYTTITSYTIPGPALW

When variants are generated, it should be noted that appropriateresidues to modify depend on the properties that are being sought insuch a variant. In the case that a variant having the sameoxidohydrolytic activity as the native parent molecule is being sought,the residues are in general those residues that are not involved in thecatalytic reaction or interaction of the enzyme with the chitinsubstrate. However, those residues may be targetted, in the alternative,to develop variants with improved reactivity. This could be achieved bystandard protein engineering techniques or by techniques based on randommutagenesis followed by screening, all techniques that are well known inthe art. Attempts to improve the function of oxidohydrolytic enzymes mayinclude improving the binding and catalytic ability of the enzyme, e.g.,to act on other substrates, e.g., carbohydrate containing copolymers,e.g., protein-carbohydrate co-polymers.

A person skilled in the art will recognize the potential of using thenative proteins' framework to create variants that are optimised forother insoluble polymeric polysaccharide substrates (e.g., other formsof chitin or cellulose), or insoluble carbohydrate-containingco-polymers.

In the case of GH61 proteins as set forth in SEQ ID NOs: 1, 2 and 3 (and15-16), preferably the residues at positions 19, 86, 169, 171 and 210 ofSEQ ID NO: 1 are conserved (see Harris et al., 2010, Biochemistry49:3305-3316, in which His-1 of the mature protein appears at position19) or the corresponding residues in other GH61 proteins. Suchcorresponding residues can easily be found by sequence alignment.

In the case of CBP21, several residues have been shown to be importantin the binding of CBP21 to chitin and more specifically to the abilityof CBP21 to enhance the degradation of chitin (Vaaje-Kolstad et al.,2005, J. Biol. Chem. 280: 11313-11319 and 28492-28497). Severalmutations have been shown not to affect binding, but to affect theability of CBP21 to enhance the degradation of chitin. These results maybe predicted for other CBM33 proteins such as EfCBM33, E7, CelS2,Cfla_0175, Cfla_0172, Cfla_0316, Cfla_0490, CJA_2191 (Cbp33A), CJA_3139(Cbp33/10B) and SC01734. These residues are preferably not modifiedrelative to the wild type CBP21 sequence as set out in SEQ ID NO: 4 (orany one of SEQ ID NOs: 5 to 14, e.g., SEQ ID NO: 5), if the aim is tomodify, e.g., the stability of the CBP (for example under processconditions), but these residues may be targeted if one's aim is toimprove or change the CBP21's functional properties.

Preferred variants of CBP21 retain one or more and preferably all of: atyrosine residue at position 54, a glutamic acid residue at position 55,a glutamic acid residue at position 60, a histidine residue at position114, an aspartic acid residue at position 182 and an asparagine atposition 185 (sequence numbering according to SEQ ID NO: 4).

In connection with amino acid sequences, “sequence similarity”,preferably “sequence identity”, refers to sequences which have thestated value when assessed using, e.g., using the SWISS-PROT proteinsequence databank using FASTA pep-cmp with a variable pamfactor and gapcreation penalty set at 12.0 and gap extension penalty set at 4.0 and awindow of 2 amino acids). Sequence identity at a particular residue isintended to include identical residues which have simply beenderivatized. Sequence identity assessments are made with reference tothe full length sequence of the recited sequence used for comparison.

Preferred “variants” include those in which instead of the naturallyoccurring amino acid the amino acid which appears in the sequence is astructural, e.g., non-native analogue thereof. Amino acids used in thesequences may also be derivatized or modified, e.g., labelled,glycosylated or methylated, providing the function of theoxidohydrolytic enzyme is not significantly adversely affected.

Further preferred variants are those in which relative to the abovedescribed amino acid sequences, the amino acid sequence has beenmodified by single or multiple amino acid (e.g., at 1 to 10, e.g., 1 to5, preferably 1 or 2 residues) substitution, addition and/or deletion orchemical modification, including deglycosylation or glycosylation, butwhich nonetheless retain functional activity, insofar as they bind tothe polysaccharide substrate and enhance its degradation, particularlywhen used in conjunction with one or more saccharolytic enzymes.

Within the meaning of “addition” variants are included amino and/orcarboxyl terminal fusion proteins or polypeptides, comprising anadditional protein or polypeptide or other molecule fused to the enzymesequence. Carboxyl terminal fusions are preferred. It must be ensuredthat any such fusion to the enzyme does not adversely affect thefunctional properties required for use in the methods of the inventionas set out elsewhere herein.

“Substitution” variants preferably involve the replacement of one ormore amino acids with the same number of amino acids and makingconservative substitutions.

Such functionally-equivalent variants mentioned above include inparticular naturally occurring biological variations (e.g., found inother microbial species) and derivatives prepared using knowntechniques. In particular functionally equivalent variants of theoxidohydrolytic enzymes described herein extend to enzymes which arefunctional in (or present in), or derived from different genera orspecies than the specific molecules mentioned herein.

Variants such as those described above can be generated in anyappropriate manner using techniques which are known and described in theart, for example using standard recombinant DNA technology.

Preferably the variants or fragments described herein are derived fromthe native sequences set forth above, particularly those of any one ofSEQ ID NOs: 1 to 14 (e.g., SEQ ID NOs: 1-4 or 1-5) and/or 15 to 16.

As referred to herein a “reducing agent” is an element or compound in aredox (reduction-oxidation) reaction that reduces another species and inso doing becomes oxidized and is therefore the electron donor in theredox reaction. Preferably the reducing agent is non-enzymatic. In thisparticular invention, the reduced compound is oxygen which by thereduction becomes activated, enhancing the oxidohydrolytic function of,e.g., GH61 or CBM33 proteins. The reducing agent may function as anelectron donor in the enzymatic process and it is possible that electrondonation takes place via the generation of reactive oxygen species suchas O₂ ⁻. The reducing agent promotes electron donation and/or thegeneration of reactive oxygen. Preferably said reducing agent isascorbic acid, reduced glutathione or Fe(II)SO₄. Further preferredreducing agents are LiAlH₄ and NaBH₄. Other preferred reducing agentsinclude organic acids (such as succinic acid, gallic acid, coumaricacid, humic acid and ferulic acid) and reducing sugars (such as glucose,glucosamine and N-acetylglucosamine), Alternatively, lignin, whichcontains reducing groups, or fragments thereof, may be used as thereducing agent. As noted above, Fe(II)SO₄ may be used as a reducingagent and in so doing will also contribute the required divalent metalion. Whilst a single compound may provide both the reducing agent andmetal ion, it is preferred that these features are provided by differentcompounds, i.e., that the reducing agent and metal ion are separatecompounds.

More than one of such agents may be used in line with methods of theinvention and may be selected according to the substrate and conditionsused (e.g., pH and temperature). It will be appreciated that theefficacy and stability of reducing agents varies between these agentsand depends on pH. Thus the pH and reducing agent should be optimizedfor the oxidohydrolytic enzyme to be used.

Preferably said divalent metal ion is Ca, Co, Mg, Mn, Ni or Zn. In analternative embodiment the divalent metal ion is Cu. Thus, for examplesalts such as MgCl₂, ZnCl₂ or CoCl₂ (or alternatively CuCl₂) may beused.

The following description sets out conditions that can be used forperformance of the method of the invention, but it should be noted thatany appropriate conditions can be used.

Prior to contacting the polysaccharide-containing material with theoxidohydrolytic enzyme, the polysaccharide-containing material may bepre-treated.

The polysaccharide-containing material may be pre-treated, e.g., todisrupt plant cell wall components, using conventional methods known inthe art. Prior to pre-treatment, where appropriate, thepolysaccharide-containing material may be subjected to pre-soaking,wetting, or conditioning using methods known in the art. Physicalpre-treatment techniques include, for example, various types of milling,irradiation, steaming/steam explosion and hydrothermolysis; chemicalpre-treatment techniques can include dilute acid, alkaline (e.g., limepre-treatment), organic solvent (such as organosols pre-treatments),ammonia treatments (e.g., ammonia percolation (APR) and ammoniafibre/freeze explosion (AFEX)), sulfur dioxide, carbon dioxide, wetoxidation and pH-controlled hydrothermolysis; and biologicalpre-treatment techniques can involve applying lignin-solubilizingmicroorganisms (see, for example, Hsu, 1996, Pre-treatment of biomass,in “Handbook on Bioethanol: Production and Utilization”, Wyman, ed.,Taylor & Francis, Washington, D.C., 179-212; Ghosh & Singh, 1993, Adv.Appl. Microbiol. 39: 295-333; McMillan, 1994, Pretreatinglignocellulosic biomass: a review, in “Enzymatic Conversion of Biomassfor Fuels Production”, Himmel et al. eds., ACS Symposium Series 566,American Chemical Society, Washington, D.C., Chapter 15; Gong et al.,1999, Advances in Biochemical Engineering/Biotechnology, Scheper, ed.,Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Olsson &Hahn-Hagerdal, 1996, Enz. Microb. Tech. 18: 312-331; and Vallander &Eriksson, 1990, Adv. Biochem. Eng./Biotechnol. 42: 63-95). Additionalpre-treatments include ultrasound, electroporation, microwave,supercritical CO₂, supercritical H₂O and ammonia percolation.

Pre-treated corn stover is a cellulose-containing material derived fromcorn stover, e.g., by treatment with heat and dilute acid.

Following optional pre-treatment, the polysaccharide-containing material(the substrate) may be exposed to the oxidohydrolytic enzyme in vitro inany appropriate vessel, e.g., by mixing together the substrate and theenzyme in an appropriate medium (e.g., a solution, such as an aqueoussolution) or by applying the enzyme to the substrate (e.g., by applyingthe enzyme in a solution to a substrate).

In a preferred embodiment the oxidohydrolytic enzyme is present in abuffer such as a phosphate buffer, e.g., a sodium phosphate buffer, orTris buffer. Suitable concentration ranges for such a buffer are 1-100mM. The oxidohydrolytic enzyme may be provided as a purified preparation(as described hereinafter) or may be present in a composition, whereinit may be a major component, preferably comprising at least 20, 30, 40,50, 60 or 70% w/w dry weight in the composition, or it may be a minorcomponent (e.g., in a mixture with one or more saccharolytic enzymes),preferably comprising at least 1, 2, 5 or 10%, e.g., 1-5%, w/w dryweight in the composition.

The enzyme can be present in the solution at any suitable concentration,such as a concentration of 0.001-1.0 mg/ml, e.g., 0.01-0.1 mg/ml or0.05-0.5 mg/ml.

The polysaccharide substrate is present in the reaction mix at anysuitable concentration which will depend to some extent on the purity ofthe polysaccharide in the material containing it. Conveniently, however,the polysaccharide itself is present at a concentration of from 0.1 to200 mg/ml, preferably 0.2 to 20 mg/ml or 0.5 to 50 mg/ml, or morepreferably 25 to 150 mg/ml, especially preferably at least 25 mg/ml.Preferably the polysaccharide is present in the material containing thepolysaccharide to a level of >50%, e.g., >60, 70, 80 or 90%, w/w dryweight in the material.

Preferably the polysaccharide substrate is exposed to the enzyme, e.g.,by incubation together, for a period of 4, 6, 12 or 24 hours or more,such as 4-24 or 6-24 hours, e.g., 36 or 48 hours or more, or 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14 days or more. In a preferred aspect theincubation is 6-24 hours. This incubation is in general carried out ator about 50° C., although appropriate temperatures for optimizing theenhancement of polysaccharide degradation can readily be determined bythe skilled person in the art. For example, the temperature can be inthe range of 20-65° C., e.g., 30-60° C., preferably 50-55° C.

It will be appreciated that the necessary incubation times, pH,temperature, substrate and enzyme concentrations are not independent ofeach other. Thus, a large range of conditions can be envisaged, whichcan easily be evaluated. The oxidohydrolytic enzymes serve to enhancedegradation by the saccharolytic enzymes and thus may allow the use oflower concentrations of the latter or shorter reaction times.

Preferably a pH in the range of 4 to 9 is used. Preferably the pH is inthe range of 5 to 8. The preferred pH is about pH 5-6.

Where a reducing agent is used, the reducing agent is preferably addedfor the duration of the degradation reaction, though it may be addedafter that reaction has commenced and may be present only while theoxidohydrolytic enzyme is present or active. Reducing agents arepreferably added to a final concentration range of 0.1 to 100 mM,preferably 0.5 to 20 mM, especially preferably 1-5 mM. Reducing agentsmay be present in the polysaccharide substrate, e.g., lignin present ina lignocellulosic biomass, but preferably said reducing agents are addedto the reaction mix.

As with the reducing agent, the metal ion may be added at the start orduring the degradation reaction. However, it may not always be necessaryto add metal ions to the reaction mixture as some substrate-containingmaterial may contain sufficient metal ions for the reaction to proceedsuccessfully. However, in a preferred aspect, metal ions are added tothe reaction mix. In one embodiment, the metal ion may be added to theoxidohydrolytic enzyme during its production or isolation or to theenzyme prior to its addition to the reaction mix such that the enzyme is“pre-loaded” with the relevant metal ion. Metal ions are preferablyadded to a final concentration range of 0.001 to 50 mM, e.g., 0.01 to 50mM, preferably 0.1 to 5 mM. The concentration to be used may be readilydetermined for the particular metal ion to be used in the reaction mix.For example, lower concentrations of Cu²⁺ (e.g., 0.001 to 0.1 mM) may beappropriate relative to the concentration required for other metal ions.It will be appreciated that the optimal metal ion concentrations to someextent depend on the enzyme and substrate concentrations used in theenzymatic conversion reactions that are set up.

As noted herein, the oxidohydrolytic enzymes are believed to catalyzehydrolysis by oxidation by molecular oxygen. It is therefore imperative(as noted in the Examples) that molecular oxygen is available for use inthe reaction. As such any conditions that result in an oxygen-free(anaerobic) environment must be avoided.

Thus, in a preferred aspect the method comprises contacting saidpolysaccharide with an oxidohydrolytic enzyme and adding at least onereducing agent and preferably at least one divalent metal ion to thereaction mixture.

Preferably the incubation is carried out with agitation, particularlywhen a cellulose-containing material is used.

In a preferred aspect, the oxidohydrolytic enzyme is used at aconcentration of 0.01 to 0.5 mg/ml and the polysaccharide substrate at25 to 150 mg/ml (when calculated according to the target substratecontent and not taking into account the additional material that may bepresent with the substrate) and the reaction is conducted at pH 5-8 for6 to 24 hours at 50 to 55° C.

In methods in which the degradation or hydrolysis is carried out withthe oxidohydrolytic enzyme only, the result of said reaction isincomplete degradation of the polysaccharide to yield largely insolublelong oligosaccharides and minor fractions of soluble oligosaccharides,perhaps including very minor fractions of disaccharides. Preferably saiddegradation or hydrolysis is enhanced further or completed by the use ofappropriate additional degradative glycoside hydrolases.

Thus in a further preferred aspect the present invention provides amethod of degrading or hydrolyzing a polysaccharide comprising:

a) contacting said polysaccharide with one or more oxidohydrolyticenzymes, wherein said degradation or hydrolysis is carried out in thepresence of at least one reducing agent and at least one divalent metalion, and

b) contacting said polysaccharide (or the degradation or hydrolysisproduct thereof) with one or more saccharolytic enzymes selected from acellulose hydrolase or chitin hydrolase.

Clearly in performing the method the oxidohydrolytic enzyme and thesaccharolytic enzyme must be selected in accordance with thepolysaccharide substrate, e.g., GH61 and a cellulose hydrolase forcellulose and CBP21 or another CBM33 family protein and a chitinhydrolase for chitin. Cross-reaction between different substrates mayalso be possible, e.g., CBM33 family proteins may be effective asoxidohydrolytic enzymes on cellulose, e.g., SEQ ID NO: 5 (EfCBM33) andother CBM33 proteins described herein may be used in methods of theinvention performed on cellulose. Similarly, GH61 family members may beused on substrates other than cellulose, e.g., chitin or hemicellulose.

Until now enzyme activity of members of the CBM33 family has only beenreported for chitin as a substrate (e.g., CBP21 from Serratiamarcescens). However, the present invention demonstrates that members ofthe CBM33 family, e.g., E7 and CelS2, work on cellulose. The enzymaticfunction entails hydrolysis (cleavage) and oxidation of cellulose chainsin insoluble cellulose crystals, which enables a more rapiddeconstruction of the cellulose by cellulases. CBM33 enzymes acting oncellulose work optimally in the presence of an external electron donor(e.g., a reducing agent) and divalent metal ions. These enzymatic traitsare highly similar to those observed previously for CBM33 enzymes thatact on chitin. In the experiments described herein a single domain CBM33from Thermobifida fusca (Uniprot ID:Q47QG3; E7) and a multidomain CBM33(Uniprot ID: Q9RJY2; a CBM33 with a CBM2 attached on the C-terminal sideof the protein; CelS2) from Streptomyces coelicolor A3(2) were expressedand purified and their ability to potentate cellulose degrading enzymeactivity was observed.

CBM33 proteins for reaction with cellulose are preferably obtained fromcellulolytic bacteria, e.g., bacteria of the genera Cellulomonas,Cellvibrio, Thermobifida or Streptomyces, e.g., bacteria of the speciesCelllulomonas flavigena, Cellvibrio japonicus, Thermobifida fusca orStreptomyces spp. (preferably E7 and CelS2 as disclosed herein andCfla_0175, Cfla_0172, Cfla_0316, Cfla_0490, CJA_2191 (Cbp33A), CJA_3139(Cbp33/10B) and SCO1734) and/or have one or more cellulose bindingmodules (e.g., belonging to CBM family 2) attached to the C-terminalend.

Natural or engineered variants of these oxidohydrolytic enzymes withaltered substrate specificity (e.g., from chitin to cellulose) may becombined with other substrates and saccharolytic enzymes.

It will be obvious to the expert in the field that polysaccharides suchas chitin and, especially, cellulose may occur in complex co-polymericmatrices including for example hemicelluloses in the case of plant cellwall material. Since cellulose and hemicelluloses interact strongly, itis possible that loosening of the cellulose structure by anoxidohydrolase may make not only the cellulose but also thehemicellulose more accessible for attack by appropriate saccharolyticenzymes. Thus, oxidohydrolases such as GH61 and CBM33 family proteinsmay also be used concomitantly with, e.g., hemicellulases or otherenzymes targeting the non-chitin and non-cellulose polymers in complexchitin- or cellulose-containing co-polymeric materials, in order toincrease the saccharolytic efficiency of these enzymes.

As referred to herein a “saccharolytic enzyme” is an enzyme which iscapable of cleaving glycosidic bonds between saccharide monomers ordimers in a polysaccharide, using a standard hydrolytic mechanism asemployed by most enzymes classified in the glycoside hydrolase (GH)families in the CAZy database. These enzymes include cellulosehydrolases, chitin hydrolases and beta-glucosidases.

As referred to herein a “cellulose hydrolase” is an enzyme whichhydrolyses cellulose or intermediate breakdown products. Preferably thehydrolase is a cellulase. Cellulases are classified as glycosylhydrolases (GH) in families based on their degree of identity and fallwithin the GH families 1, 3, 5-9, 12, 44, 45, 48 and 74. Based onmechanism they can be grouped into exo-1,4-beta-D-glucanases orcellobiohydrolases (CBHs, EC 3.2.1.91), endo-1,4-beta-D-glucanases (EGs,EC 3.2.1.4) and beta-glucosidases (BGs, EC 3.2.1.21). EGs cleaveglycosidic bonds within cellulose microfibrils, acting preferentially atamorphous cellulose regions. EGs fragment cellulose chains to generatereactive ends for CBHs, which act “processively” to degrade cellulose,including crystalline cellulose, from either the reducing (CBH1) ornon-reducing (CBHII) ends, to generate mainly cellobiose. Cellobiose isa water-soluble beta-1,4-linked dimer of glucose. Beta-glucosidaseshydrolyze cellobiose to glucose. GH61 enzymes were previously classifiedas weak endoglucanases on the basis of activity of one member of thefamily though this is now not considered correct. As mentioned above,GH61 enzymes have been found to be oxidohydrolytic by the presentinventors.

The ability of cellulose hydrolases to hydrolyse cellulose may beassessed by using methods known in the art, including methods in whichnon-modified cellulose is used as substrate. Activity is then measuredby measuring released products, using either HPLC-based methods ormethods that determine the number of newly formed reducing ends (e.g.,Zhang et al., 2009, Methods Mol. Biol. 581:213-31; Zhang et al., 2006,Biotechnol. Adv. 24(5): 452-81). In the alternative, the efficacy of thecellulose hydrolase may be assessed by using an appropriate substrateand determining whether the viscosity of the incubation mixturedecreases during the reaction. The resulting reduction in viscosity maybe determined by a vibration viscosimeter (e.g., MIVI 3000 fromSofraser, France). Determination of cellulase activity, measured interms of Cellulase Viscosity Unit (CEVU), quantifies the amount ofcatalytic activity present in a sample by measuring the ability of thesample to reduce the viscosity of a solution of the substrate.

Cellulases may be obtained from commercial sources, i.e., companies suchas Novozymes, Danisco and Biocatalysts. Examples of commercialcellulases include, for example, CELLIC™ CTec (Novozymes A/S), CELLIC™CTec2 (Novozymes A/S), CELLUCLAST™ (Novozymes A/S), NOVOZYM™ 188(Novozymes A/S), CELLUZYME™ (Novozymes A/S), CEREFLO™ (Novozymes A/S),and ULTRAFLO™ (Novozymes A/S), ACCELERASE™ (Genencor Int.), LAMINEX™(Genencor Int.), SPEZYME™ CP (Genencor Int.), FILTRASE® NL (DSM);METHAPLUS® S/L 100 (DSM), ROHAMENT™ 7069 W (Röhm GmbH), FIBREZYME® LDI(Dyadic International, Inc.), FIBREZYME® LBR (Dyadic International,Inc.), or VISCOSTAR® 150 L (Dyadic International, Inc.).

Alternatively cellulases may be produced using standard recombinanttechniques for protein expression. The scientific literature containsnumerous examples of the cloning, overexpression, purification andsubsequent application of all types of cellulases, e.g., endoglucanases,cellobiohydrolases, and beta-glucosidases.

Examples of bacterial endoglucanases that can be used in the methods ofthe present invention, include, but are not limited to, an Acidothermuscellulolyticus endoglucanase (WO 91/05039; WO 93/15186; U.S. Pat. No.5,275,944; WO 96/02551; U.S. Pat. No. 5,536,655, WO 00/70031, WO05/093050); Thermobifida fusca endoglucanase III (WO 05/093050); andThermobifida fusca endoglucanase V (WO 05/093050).

Examples of fungal endoglucanases that can be used in the presentinvention include, but are not limited to, a Trichoderma reeseiendoglucanase I (Penttila et al., 1986, Gene 45: 253-263; Trichodermareesei Cel7B endoglucanase I; GENBANK™ accession no. M15665);Trichoderma reesei endoglucanase II (Saloheimo, et al., 1988, Gene63:11-22; Trichoderma reesei Cel5A endoglucanase II; GENBANK™ accessionno. M19373); Trichoderma reesei endoglucanase III (Okada et al., 1988,Appl. Environ. Microbiol. 64: 555-563; GENBANK™ accession no. AB003694);Trichoderma reesei endoglucanase V (Saloheimo et al., 1994, MolecularMicrobiology 13: 219-228; GENBANK™ accession no. Z33381); Aspergillusaculeatus endoglucanase (Ooi et al., 1990, Nucleic Acids Research 18:5884); Aspergillus kawachii endoglucanase (Sakamoto et al., 1995,Current Genetics 27: 435-439); Erwinia carotovara endoglucanase(Saarilahti et al., 1990, Gene 90:9-14); Fusarium oxysporumendoglucanase (GENBANK™ accession no. L29381); Humicola grisea var.thermoidea endoglucanase (GENBANK™ accession no. AB003107); Melanocarpusalbomyces endoglucanase (GENBANK™ accession no. MAL515703); Neurosporacrassa endoglucanase (GENBANK™ accession no. XM_324477); Humicolainsolens endoglucanase V; Myceliophthora thermophila CBS 117.65endoglucanase; basidiomycete CBS 495.95 endoglucanase; basidiomycete CBS494.95 endoglucanase; Thielavia terrestris NRRL 8126 CEL6Bendoglucanase; Thielavia terrestris NRRL 8126 CEL6C endoglucanase;Thielavia terrestris NRRL 8126 CEL7C endoglucanase; Thielavia terrestrisNRRL 8126 CEL7E endoglucanase; Thielavia terrestris NRRL 8126 CEL7Fendoglucanase; Cladorrhinum foecundissimum ATCC 62373 CEL7Aendoglucanase; and Trichoderma reesei strain No. VTT-D-80133endoglucanase (GENBANK™ accession no. M15665).

Examples of cellobiohydrolases useful in the present invention include,but are not limited to, Trichoderma reesei cellobiohydrolase I;Trichoderma reesei cellobiohydrolase II; Humicola insolenscellobiohydrolase I; Myceliophthora thermophila cellobiohydrolase II;Thielavia terrestris cellobiohydrolase II (CEL6A); Chaetomiumthermophilum cellobiohydrolase I; and Chaetomium thermophilumcellobiohydrolase II.

Examples of beta-glucosidases useful in the present invention include,but are not limited to, Aspergillus oryzae beta-glucosidase; Aspergillusfumigatus beta-glucosidase; Penicillium brasilianum IBT 20888beta-glucosidase; Aspergillus niger beta-glucosidase; and Aspergillusaculeatus beta-glucosidase.

Cellulase mixtures may be used, e.g., a cellulase mixture whichcomprises at least one endoglucanase, e.g., belonging to GH family 5, 7or 12, a cellobiohydrolase moving towards the reducing end, e.g.,belonging to GH family 6, a cellobiohydrolase moving towards thenon-reducing end, e.g., belonging to GH family 7, and abeta-glucosidase. More preferably, more complex mixtures are used, inparticular mixtures containing several endoglucanases with differentsubstrate specificities (e.g., acting at different faces of thecellulose crystals). Appropriate cellulases may be readily identifiedtaking into account the substrate to be degraded.

As referred to herein a “chitin hydrolase” is an enzyme which hydrolyseschitin or intermediate breakdown products. Preferably said chitinhydrolase is a chitinase, chitosanase or lysozyme. The degradation maybe complete or partial. For example, the activity of some chitinhydrolase, e.g., chitinases on chitin substrates is not strong enough toresult in complete degradation of the substrate. This is particularlythe case for chitinases such as ChiG from Streptomyces coelicolor thatdo not have their own CBM, or chitinases such as ChiB from S.marcescens. In this case, the use of a oxidohydrolytic enzyme that actson chitin in accordance with the present invention can result inenhanced chitin degradation and preferentially result in completedegradation that was not previously possible. Other chitinases, such asChiC from S. marcescens, are capable of completely degrading chitin, butthe speed of this process increases upon addition of an oxidohydrolyticenzyme such as CBP21.

Chitinase enzymes are found in plants, microorganisms and animals.Chitinases have been cloned from various species of microorganisms andhave been categorised into two distinct families, designated family GH18and family GH19 of the glycoside hydrolases, based on sequencesimilarities (Henrissat and Bairoch, 1993, Biochem, J. 293:781-788).These enzymes are referred to collectively herein as chitin hydrolases.

There are several ways to measure chitinase activity that are well knownin the field, including methods in which non-modified chitin is used assubstrate. Activity on non-modified chitin is measured by measuringreleased products, using either HPLC-based methods or methods thatdetermine the number of newly formed reducing ends.

Chitinases may be obtained from commercial sources, i.e., companies suchas Sigma. Alternatively chitinases may be produced using standardrecombinant techniques for protein expression. The scientific literaturecontains numerous examples of the cloning, overexpression, purificationand subsequent application of all types of chitinases (e.g., Horn etal., 2006, FEBS J. 273(3):491-503 and references therein).

Other suitable hydrolytic enzymes for hydrolyzing additionalnon-cellulose (or non-chitin) polysaccharides include hemicellulasessuch as acetylxylan esterases, arabinofurosidases, feruloyl esterases,glucuronidases, mannanases, xylanases, and xylosidases.

The cellulase mixtures may also be used in conjunction withhemicellulases. Hemicellulases may also be obtained from commercialsources. Examples of commercial hemicellulases include, for example,SHEARZYME™ (Novozymes A/S), CELLIC™ HTec (Novozymes A/S), CELLIC™ HTec2(Novozymes A/S), VISCOZYME® (Novozymes A/S), ULTRAFLO® (Novozymes A/S),PULPZYME® HC (Novozymes A/S), MULTIFECT® Xylanase (Genencor),ACCELLERASE® XY (Genencor), ACCELLERASE® XC (Genencor), ECOPULP® TX-200A(AB Enzymes), HSP 6000 Xylanase (DSM), DEPOL™ 333P (BiocatalystsLimited, Wales, UK), DEPOL™ 740 L. (Biocatalysts Limited, Wales, UK),and DEPOL™ 762P (Biocatalysts Limited, Wales, UK).

Examples of xylanases useful in the methods of the present inventioninclude, but are not limited to, Aspergillus aculeatus xylanase(GeneSeqP:AAR63790; WO 94/21785), Aspergillus fumigatus xylanases (WO2006/078256), and Thielavia terrestris NRRL 8126 xylanases (WO2009/079210).

Examples of beta-xylosidases useful in the methods of the presentinvention include, but are not limited to, Trichoderma reeseibeta-xylosidase (UniProtKB/TrEMBL accession number Q92458), Talaromycesemersonii (SwissProt accession number Q8X212), and Neurospora crassa(SwissProt accession number Q7SOW4).

Examples of acetylxylan esterases useful in the methods of the presentinvention include, but are not limited to, Hypocrea jecorina acetylxylanesterase (WO 2005/001036), Neurospora crassa acetylxylan esterase(UniProt accession number q7s259), Thielavia terrestris NRRL 8126acetylxylan esterase (WO 2009/042846), Chaetomium globosum acetylxylanesterase (Uniprot accession number Q2GWX4), Chaetomium gracileacetylxylan esterase (GeneSeqP accession number AAB82124), Phaeosphaerianodorum acetylxylan esterase (Uniprot accession number Q0UHJ1), andHumicola insolens DSM 1800 acetylxylan esterase (WO 2009/073709).

Examples of ferulic acid esterases useful in the methods of the presentinvention include, but are not limited to, Humicola insolens DSM 1800feruloyl esterase (WO 2009/076122), Neurospora crassa feruloyl esterase(UniProt accession number Q9HGR3), and Neosartorya fischeri feruloylesterase (UniProt Accession number A1D9T4).

Examples of arabinofuranosidases useful in the methods of the presentinvention include, but are not limited to, Humicola insolens DSM 1800arabinofuranosidase (WO 2009/073383) and Aspergillus nigerarabinofuranosidase (GeneSeqP accession number AAR94170).

Examples of alpha-glucuronidases useful in the methods of the presentinvention include, but are not limited to, Aspergillus clavatusalpha-glucuronidase (UniProt accession number alcc12), Trichodermareesei alpha-glucuronidase (Uniprot accession number Q99024),Talaromyces emersonii alpha-glucuronidase (UniProt accession numberQ8X211), Aspergillus niger alpha-glucuronidase (Uniprot accession numberQ96WX9), Aspergillus terreus alpha-glucuronidase (SwissProt accessionnumber Q0CJP9), and Aspergillus fumigatus alpha-glucuronidase (SwissProtaccession number Q4VWV45).

Whilst the use of native saccharolytic enzymes is preferred, variantsdefined in accordance with the properties described herein for theoxidohydrolytic enzyme's variants may also be used.

Preferably, when said polysaccharide is cellulose, said saccharolyticenzyme is an endo-1,4-beta-D-glucanase optionally used in combinationwith other 1,4-beta-D-glucanases such as cellobiohydrolases and/orbeta-glucosidases.

Thus, the enzymes to be used in methods of the invention may be selectedbased on the polysaccharide substrate to be hydrolyzed. For example,CBP21 binds only to beta-chitin and would therefore be an appropriateoxidohydrolytic enzyme to use if the methods of the invention were to beapplied to beta-chitin. Similarly ChbB from B amyloliquifaciens asdescribed in Chu et al. (supra) may be applied to beta-chitin.

CHB1, CHB2 and CHB3 have all been isolated from S olivaceovirides(Svergun et al., Zeltins et al., Schnellman et al., Kolbe et al., Saitoet al., supra). The binding preferences of these three proteins havebeen determined and CHB1 and CHB2 bind preferably to alpha-chitin,whereas CHB3 binds to both alpha- and beta-chitin. CBP1 from Alteromonass described by Tsujibo et al. binds to both alpha- and beta-chitin, witha preference for the alpha form.

Alternatively, GH61 family members, such as described herein, may beused to assist with chitin degradation in methods of the invention.

Similarly when the substrate is chitin, the saccharolytic enzyme can beselected accordingly. The properties of chitinases have been documented(e.g., Hollis et al., 1997, Arch. Biochem. Biophys. 344: 335-342 andSuzuki et al., 1998, Biosci. Biotech. Bioch. 62: 128-135; Horn et al.,2006).

Preferred combinations for beta-chitin hydrolysis are CBP21 (or variantsor fragments thereof) with one or more of ChiA, ChiB, ChiC and ChiG.Preferred combinations for alpha-chitin hydrolysis are CHB1 or CHB2 (orvariants or fragments thereof) with one or more of ChiA, ChiB, ChiC andChiG. Alternatively GH61 family members as described herein may be usedwith appropriate chitinases.

When the substrate is cellulose, the oxidohydrolytic enzyme ispreferably a GH61 family protein (as described herein), though in viewof their ability to act on cellulose, CBM33 family proteins may also beused. Appropriate saccharolytic enzymes may be selected from knownenzymes, e.g., cellulases as described herein.

In a preferred aspect two or more oxidohydrolytic enzymes are employedin the methods of the invention. In view of their preferred substratespecificities, enhanced degradative effects may be expected when usedtogether. Thus, for example, one may use two or more CBM33 familyproteins and/or GH61 family proteins (as described herein), e.g., two ormore CBM33 family proteins, two or more GH61 family proteins or acombination of one or more of each of the CBM33 family proteins and GH61family proteins in the methods (e.g., at least one CBM33 family proteinand at least one GH61 family protein). Thus, for example chitin (orcellulose) may be contacted with one CBM33 family protein and one GH61family protein, e.g., preferably selected from the proteins describedherein (e.g., polypeptides which comprise an amino acid sequence as setforth in any one of SEQ ID NOs: 4 and/or 5 and/or 6 to 14 or relatedsequences or fragments described herein (CBM33 family proteins) andpolypeptides which comprise an amino acid sequence as set forth in anyone of SEQ ID NOs: 1 to 3 and/or 15 to 16 or related sequences orfragments described herein (GH61 family proteins)).

Appropriate enzymes for use in accordance with the invention can bedetermined by use of screening techniques to assess in vitro hydrolysis,e.g., as described in the Examples.

To identify oxidohydrolytic enzymes which may be used in combination,the enzymes may be assessed to determine whether their activity willachieve enhanced effects on the substrate. For example, when degradingbiomass one may combine members of the CBM33/GH61 families that areknown from experiments, such as those described herein, to havedifferent specifities for the various forms of chitin (e.g.,alpha-chitin or beta-chitin) or cellulose (e.g., various types ofcellulose fibers, cellulose pulps, filter paper, microcrystallinecellulose, AVICEL®, Carboxymethylcellulose) that occur in nature,biomass, and/or pretreated biomass, or can be obtained by chemicalmodification, many of these forms being easily accessible forexperimentation. In biomass, chitin and cellulose often occur asheteropolymers, containing other polysaccharides often referred to ashemicelluloses or even proteins. The different members of the CBM33/GH61families can be expected to have different activity on these differentsubstrates. Biomass is often heterogeneous, either by nature, or becausebiomasses are mixed during process development in the factory. By mixingmembers of the CBM33/GH61 families with known differences in biomasspreferences, more efficient processes may be obtained. Further synergiesmay be obtained using members of the CBM33/GH61 families thatpreferentially act on different polysaccharides in biomass, such asxylan.

Oxidohydrolytic enzymes with different activities may also be identifiedby examining the periodicity of the reaction products (see the Examplesherein). Thus, a combination may be made between oxidohydrolytic enzymes(e.g., members of the CBM33/GH61 families) with different periodicities.The periodicity for CBP21 is shown in FIGS. 2D, 6A and 7B; periodicityfor EfCBM33 is shown in FIG. 15D; periodicity for CelS2 is shown inFIGS. 17, 19, 23A, 27 and 31; and periodicity for E7 is shown in FIG.29. One possible explanation for variation in periodicity is thatcrystalline cellulose has several forms and that crystals have differentfaces (i.e., types of surface; see, e.g., Carrard et al., 2000, ProcNatl Acad Sci USA 97(19):10342-7) and that different members of theCBM33 and GH61 families attack different faces. In view of the differentactivity of the enzymes, combinations of enzymes (which may achievesynergistic effects) may be made. Thus, for example, one could combine aGH61 with CelS2.

In the methods described above using both an oxidohydrolytic enzyme anda saccharolytic enzyme, the step with the oxidohydrolytic enzyme iscarried out under conditions which allow the enzyme to interact or bindto the polysaccharide as described herein. The same conditions andconsiderations are applied to the additional step using additionalsaccharolytic enzymes (hydrolases), which step may be carried outsimultaneously or subsequent to the first step. In total the incubationmay be conducted for 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days ormore, but is typically performed for preferably about 8 to about 96hours, more preferably about 8 to about 72 hours and most preferablyabout 8 to about 48 hours or 4 to 24 hours.

Preferably aqueous solutions of the enzymes are used and preferably theenzymatic hydrolysis is carried out in a suitable aqueous environmentunder conditions that can be readily determined by one skilled in theart.

Each enzyme used in the methods may be provided as a purifiedpreparation (as described hereinafter) or may be present in acomposition, (e.g., including the other enzymes for use in the methods)preferably at at least 1, 2, 5 or 10%, preferably 1-5% w/w dry weight inthe composition.

For the methods described herein, the hydrolysis can be carried out as afed batch or continuous process where the polysaccharide-containingmaterial (substrate), which may be pre-treated, is fed gradually to, forexample, an enzyme containing hydrolysis solution.

The saccharification is generally performed in stirred-tank reactors orfermentors under controlled pH, temperature and mixing conditions asdiscussed herein. Suitable process time, temperature and pH conditionscan readily be determined by one skilled in the art and are discussedherein and can depend on the substrate and enzymes used and theirconcentrations and whether the substrate has been pretreated and whethera fermenting organism is included, see hereinbelow.

The dry solids content is in the range of preferably about 5 to about 50wt. %, more preferably about 10 to about 40 wt. % and most preferablyabout 15 to about 30 wt. %.

Each enzyme used in the reaction can be present in the solution at anysuitable concentration, such as a concentration of 0.001-1.0 mg/ml,e.g., 0.01-0.1 mg/ml or 0.05-0.5 mg/ml. Alternatively expressed, theenzymes may be used at a concentration of 0.1-100 mg enzyme/g ofpolysaccharide substrate, e.g., 1-50 mg/g substrate. Suitableconcentrations can be determined depending on the substrate and thematerial containing the substrate and the conditions of the reaction,e.g., temperature, pH and duration.

The steps in which the oxidohydrolytic enzyme and the saccharolyticenzyme(s) are contacted with the polysaccharide substrate may beperformed separately or together or a combination thereof, e.g., theoxidohydrolytic enzyme may be added and after an initial incubationperiod the saccharolytic enzyme(s) may be added. In the alternative, theoxidohydrolytic enzyme may be removed before the saccharolytic enzyme isadded. Any steps in which the oxidohydrolytic enzyme is not present(e.g., a step in which only a saccharolytic enzyme is used) need not beconducted in the presence of a reducing agent and/or metal ion.

Other enzymes may also be added in addition to or as an alternative tothe chitin or cellulose hydrolytic enzymes discussed above, depending onthe nature of the substrate that is to be degraded. For example, if thepolysaccharide to be degraded is a copolymer which contains protein,proteases may also be added. Suitable examples include Alcalase,Neutrase, Papain and other broad-specificity proteolytic enzymes. Ineach experimental set-up the suitability of proteases will need to bechecked, especially if other enzymes (e.g., chitinases or cellulases),which may be destroyed by some of the available proteases, are presentsimultaneously. If the polysaccharide is a copolymer withhemicelluloses, hemicellulolytic enzymes may be added.

Furthermore, the product resulting from using the above describedoxidohydrolytic enzyme and saccharolytic enzymes may include solubleshort oligosaccharides (particularly disaccharides). Since dimericproducts inhibit glycoside hydrolases and since monomers are the mostdesirable product resulting from the degradation/hydrolysis process forfurther processing (see hereinbelow), additional enzymes, namelybeta-glucosidases are preferably also used in the methods of theinvention.

Thus in a preferred aspect, said method of degrading or hydrolyzing apolysaccharide further comprising contacting said polysaccharide (or thedegradation or hydrolysis product thereof) with one or morebeta-glucosidases. Such enzymes may be identified and used as specifiedherein (e.g., in relation to their concentration) for othersaccharolytic enzymes. For cellulose a beta-glucosidase(s) may be usedand for chitin a beta-N-acetylglucosaminidase(s) may be used.

The steps in which the oxidohydrolytic enzyme, saccharolytic enzyme(s)and beta-glucosidase(s) are contacted with the polysaccharide substratemay be performed separately or together or a combination thereof, e.g.,the oxidohydrolytic enzyme may be added and after an initial incubationperiod the saccharolytic enzyme(s) and beta-glucosidase(s) may be added,or the latter two enzymes may be added sequentially. In the alternative,the oxidohydrolytic enzyme may be removed before the other enzymes areadded. Any steps in which the oxidohydrolytic enzyme is not present(e.g., a step in which only a saccharolytic enzyme(s) and/orbeta-glucosidase(s) is used) need not be conducted in the presence of areducing agent and/or metal ion.

The oxidohydrolytic enzymes and saccharolytic enzymes for use in themethods of the invention may be isolated, extracted or purified fromvarious different sources or synthesised by various different means. Asmentioned above the enzymes may be provided in purified preparations orin the presence of other components.

Chemical syntheses may be performed by methods well known in the artinvolving, in the case of peptides, cyclic sets of reactions ofselection deprotection of the functional groups of a terminal amino acidand coupling of selectively protected amino acid residues, followedfinally by complete deprotection of all functional groups. Synthesis maybe performed in solution or on a solid support using suitable solidphases known in the art, such as the well known Merrifield solid phasesynthesis procedure.

Preferably the enzymes for use in the invention are substantiallypurified, e.g., pyrogen-free, e.g., more than 70%, especially preferablymore than 90% pure (as assessed for example, in the case of peptides orproteins, by an appropriate technique such as peptide mapping,sequencing or chromatography or gel electrophoresis). Purification maybe performed for example by chromatography (e.g., HPLC, size-exclusion,ion-exchange, affinity, hydrophobic interaction, reverse-phase) orcapillary electrophoresis.

Recombinant expression of proteins is also well known in the art and anappropriate nucleic acid sequence can be used to express the enzymesused herein for subsequent expression and optional purification usingtechniques that are well known in the art. For example, an appropriatenucleic acid sequence can be operably linked to a promoter forexpression of the enzyme to be used in bacterial cells, e.g., E. coliwhich may then be isolated or if the enzyme is secreted, the culturemedium or the host expressing the enzyme may be used as the source ofthe enzyme.

The methods described above have applications in a number of differentfields in which hydrolysis of polysaccharides forms one of the methodsteps or in which the products of that hydrolysis are useful.

Thus in a further aspect the present invention provides a method ofproducing soluble saccharides, wherein said method comprises degradingor hydrolyzing a polysaccharide by contacting said polysaccharide withone or more oxidohydrolytic enzymes, wherein said degradation orhydrolysis is carried out in the presence of at least one reducing agentand at least one divalent metal ion and said degradation or hydrolysisreleases said soluble saccharides.

In an alternative preferred aspect the invention provides a method ofproducing soluble saccharides, wherein said method comprises degradingor hydrolyzing a polysaccharide by:

a) contacting said polysaccharide with one or more oxidohydrolyticenzymes, wherein said degradation or hydrolysis is carried out in thepresence of at least one reducing agent and at least one divalent metalion,

b) contacting said polysaccharide (or the degradation or hydrolysisproduct thereof) with one or more saccharolytic enzymes selected from acellulose hydrolase or chitin hydrolase, and optionally

c) contacting said polysaccharide (or the degradation or hydrolysisproduct thereof) with one or more beta-glucosidases;

wherein said degradation or hydrolysis releases said solublesaccharides.

The result of complete hydrolysis is soluble sugars. Usually, a mixtureof monomeric sugars and higher order oligosaccharides (e.g.,disaccharides) are generated. As discussed above, preferablybeta-glucosidases are used to produce monomeric sugars. The partially orcompleted degraded polysaccharide-containing material is preferablyrecovered for further processing, e.g., fermentation. Soluble productsof degradation of the polysaccharide-containing material can beseparated from the insoluble material using technology well known in theart such as centrifugation, filtration and gravity settling.

Preferably said soluble saccharides are isolated or recovered after saiddegradation or hydrolysis process. Preferably the soluble saccharideswhich are isolated or recovered are chitobiose and/orN-acetylglucosamine (from chitin) or cellobiose and/or glucose (fromcellulose) and/or oligosaccharides thereof.

N-acetylglucosamine and oligosaccharides of N-acetylglucosamine have anumber of commercial uses including use as a food supplement. Chitinfragments have found utility in various applications including use asimmune stimulants (Aam et al., 2010, Drugs 8(5): 1482-517).

The soluble saccharides resulting from hydrolysis of cellulose havevarious applications, particularly for use as a source of energy infermentation reactions.

Preferably the saccharide mixture released after hydrolysis containingmonomeric sugars is fermented to generate an organic substance such asan alcohol, e.g., ethanol.

Thus the present invention further provides a method of producing anorganic substance, preferably an alcohol, comprising the steps of:

i) degrading or hydrolyzing a polysaccharide by a method comprising:

-   -   a) contacting said polysaccharide with one or more        oxidohydrolytic enzymes, wherein said degradation or hydrolysis        is carried out in the presence of at least one reducing agent        and at least one divalent metal ion, and    -   b) contacting said polysaccharide (or the degradation or        hydrolysis product thereof) with one or more saccharolytic        enzymes selected from a cellulase or chitinase, and optionally    -   c) contacting said polysaccharide (or the degradation or        hydrolysis product thereof) with one or more beta-glucosidases;

to produce a solution comprising soluble saccharides;

ii) fermenting said soluble saccharides, preferably with one or morefermenting microorganisms to produce said organic substance as thefermentation product; and optionally

iii) recovering said organic substance.

Optionally, said soluble saccharides produced in step (i) may beisolated or purified from said solution.

The organic substance thus produced forms a further aspect of theinvention.

As referred to herein “soluble saccharides” include monosaccharides,disaccharides and oligonucleotides which are water soluble, preferablymono- and/or disaccharides. Preferably said soluble saccharides arefermentable, e.g., glucose, xylose, xylulose, arabinose, maltose,mannose, galactose and/or soluble oligosaccharides.

“Fermentation” refers to any fermentation process or any processcomprising a fermentation step.

The above method may additionally comprise the use of one or moreadditional enzymes such as esterases (e.g., lipases, phospholipasesand/or cutinases), proteases, laccases and peroxidases.

The steps of hydrolysis (saccharification) and fermentation may beperformed separately and/or simultaneously and include, but are notlimited to, separate hydrolysis and fermentation (SHF), simultaneoussaccharification and fermentation (SSF), simultaneous saccharificationand cofermentation (SSCF), hybrid hydrolysis and fermentation (HHF),separate hydrolysis and co-fermentation (SHCF), hybrid hydrolysis andcofermentation (HHCF) and direct microbial conversion (DMC).Conveniently, any method known in the art comprising pre-treatment,enzymatic hydrolysis (saccharification), fermentation, or a combinationthereof, can be used in the practicing the above methods.

Conveniently, a conventional apparatus can include a fed-batch stirredreactor, a batch stirred reactor, a continuous flow stirred reactor withultrafiltration and/or a continuous plug-flow column reactor (deCastilhos Corazza et al., 2003, Acta Scientiarum. Technology 25: 33-38;Gusakov & Sinitsyn, 1985, Enz. Microb. Technol. 7: 346-352), anattrition reactor (Ryu & Lee, 1983, Biotechnol. Bioeng. 25: 53-65), or areactor with intensive stirring induced by an electromagnetic field(Gusakov et al., 1996, Appl. Biochem. Biotechnol. 56: 141-153).Additional reactor types include, for example, fluidized bed, upflowblanket, immobilized and extruder type reactors for hydrolysis and/orfermentation.

Pre-treatments that may be used were discussed herein and apply to allmethods of the invention. The polysaccharide-containing material can bepre-treated before hydrolysis and/or fermentation. Pre-treatment ispreferably performed prior to the hydrolysis step. Alternatively, thepretreatment can be carried out simultaneously with hydrolysis, such assimultaneously with treatment of the polysaccharide-containing materialwith the enzymes used in the methods (i.e., oxidohydrolytic andsaccharolytic enzymes) to release fermentable sugars, such as glucoseand/or cellobiose. In most cases the pre-treatment step itself resultsin some conversion of biomass to fermentable sugars (even in the absenceof enzymes).

The fermentable sugars obtained by the method of the invention can befermented by one or more fermenting microorganisms capable of fermentingthe sugars directly or indirectly into a desired fermentation product.

The fermentation conditions depend on the desired fermentation productand fermenting organism and can easily be determined by one skilled inthe art.

In the fermentation step, sugars, released from the substrate arefermented to a product, e.g., ethanol, by a fermenting organism, such asyeast. The polysaccharide substrate to be used in the method may beselected based on the desired fermentation product.

The “fermenting microorganism” refers to any microorganism, includingbacterial and fungal organisms, suitable for use in the fermentationprocess to produce a fermentation product. The fermenting organism canbe C6 and/or C5 fermenting organisms, or a combination thereof. Both C6and C5 fermenting organisms are well known in the art. Suitablefermenting microorganisms are able to ferment, i.e., convert, sugars,such as glucose, xylose, xylulose, arabinose, maltose, mannose,galactose, or oligosaccharides, directly or indirectly into the desiredfermentation product.

Examples of bacterial and fungal fermenting organisms producing ethanolare described by Lin et al., 2006, Appl. Microbiol. Biotechnol. 69:627-642.

Examples of fermenting microorganisms that can ferment C6 sugars includebacterial and fungal organisms, such as yeast. Preferred yeast includesstrains of Saccharomyces spp., preferably Saccharomyces cerevisiae.

Examples of fermenting organisms that can ferment C5 sugars includebacterial and fungal organisms, such as yeast. Preferred C5 fermentingyeast include strains of Pichia, preferably Pichia stipitis, such asPichia stipitis CBS 5773; strains of Candida, preferably Candidaboidinii, Candida brassicae, Candida sheatae, Candida diddensii, Candidapseudotropicalis or Candida utilis.

Other fermenting organisms include strains of Zymomonas, such asZymomonas mobilis; Hansenula, such as Hansenula anomala; Klyveromyces,such as K. fragilis; Schizosaccharomyces, such as S. pombe; and E. coli,especially E. coli strains that have been genetically modified toimprove the yield of ethanol.

In a preferred aspect, the yeast is a Saccharomyces spp. In a morepreferred aspect, the yeast is Saccharomyces cerevisiae, Saccharomycesdistaticus, Saccharomyces uvarum. In another preferred aspect, the yeastis a Kluyveromyces, e.g., Kluyveromyces marxianus or Kluyveromycesfragilis.

Other yeast that may be used include Clavispora, e.g., Clavisporalusitaniae or Clavispora opuntiae; Pachysolen, e.g., Pachysolentannophilus; and Bretannomyces, e.g., Bretannomyces clausenii.

Bacteria that can efficiently ferment hexose and pentose to ethanolinclude, for example, Zymomonas, such as Zymomonas mobilis andClostridium, such as Clostridium thermocellum.

Commercially available yeast suitable for ethanol production include,e.g., ETHANOL RED™ yeast (available from Fermentis/Lesaffre, USA), FALI™(available from Fleischmann's Yeast, USA), SUPERSTART™ and THERMOSACC™fresh yeast (available from Ethanol Technology, Wis., USA), BIOFERM™ AFTand XR (available from NABC—North American Bioproducts Corporation, Ga.,USA), GERT STRAND™ (available from Gert Strand AB, Sweden) and FERMIOL™(available from DSM Specialties).

The fermenting microorganism(s) is typically added to the degradedpolysaccharide-material and the fermentation is performed for about 8 toabout 96 hours, such as about 24 to about 60 hours. The temperature istypically between about 26° C. to about 60° C., in particular about 32°C. to 50° C. and at about pH 3 to about pH 8, such as around pH 4-5, 6,or 7. The above conditions will of course depend on various factorsincluding the fermenting microorganism that is used.

The fermenting microorganism(s) is preferably applied in amounts ofapproximately 10⁵ to 10¹², preferably from approximately 10⁷ to 10¹⁰,especially approximately 2×10⁸ viable cell count per ml of fermentationbroth.

For ethanol production, following the fermentation the fermented slurryis distilled to extract the ethanol. The ethanol obtained according tothe methods of the invention can be used as, e.g., fuel ethanol,drinking ethanol, i.e., potable neutral spirits, or industrial ethanol.

A fermentation stimulator can be used in combination with any of theenzymatic processes described herein to further improve the fermentationprocess, and in particular, the performance of the fermentingmicroorganism, such as, rate enhancement and ethanol yield. A“fermentation stimulator” refers to stimulators for growth of thefermenting microorganisms, in particular, yeast. Preferred fermentationstimulators for growth include vitamins and minerals. Examples ofvitamins include multivitamins, biotin, pantothenate, nicotinic acid,meso-inositol, thiamine, pyridoxine, para-aminobenzoic acid, folic acid,riboflavin and Vitamins A, B, C, D and E.

The organic substance which is the fermentation product can be anysubstance derived from the fermentation. The fermentation product canbe, without limitation, an alcohol (e.g., arabinitol, butanol, ethanol,glycerol, methanol, 1,3-propanediol, sorbitol or xylitol); an organicacid (e.g., acetic acid, acetonic acid, adipic acid, ascorbic acid,citric acid, 2,5-diketo-D-gluconic acid, formic acid, fumaric acid,glucaric acid, gluconic acid, glucuronic acid, glutaric acid,3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid, malonicacid, oxalic acid, propionic acid, succinic acid or xylonic acid); aketone (e.g., acetone); an aldehyde (e.g., formaldehyde); an amino acid(e.g., aspartic acid, glutamic acid, glycine, lysine, serine orthreonine); or a gas (e.g., methane, hydrogen (H₂), carbon dioxide (CO₂)or carbon monoxide (CO)). The fermentation product may also be analkane, a cycloalkane, an alkene, isoprene, or polyketide. Thefermentation product can also be protein.

In a preferred aspect, the fermentation product is an alcohol. It willbe understood that the term “alcohol” encompasses a substance thatcontains one or more hydroxyl moieties. Preferably the alcohol isarabinitol, butanol, ethanol, glycerol, methanol, 1,3-propanediol,sorbitol or xylitol. Ethanol is the preferred product.

The fermentation product(s) may be recovered from the fermentationmedium using any method known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), distillation or extraction. Forexample, ethanol is separated from the fermented cellulose-containingmaterial and purified by conventional methods of distillation. Ethanolwith a purity of up to about 96 vol. % can be obtained.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES Example 1: Effects of Oxidohydrolytic Enzymes CBP21, EfCBM33,CelS2 and E7 on Chitin or Cellulose Substrates

Materials and Methods

Reagents

Pure beta-chitin powder (80 #mesh) from squid pen was purchased fromFrance Chitin (Marseille, France). H₂ ¹⁸O (containing 97% ¹⁸O) and ¹⁸O₂(containing 99% ¹⁸O) was purchased from Cambridge Isotope LaboratoriesInc. (Andover, Mass.). 2,5-dihydroxy-benzoic acid (DHB) was purchasedfrom Bruker Daltonics (Bremen, Germany). Dithionite, ascorbic acid,reduced glutathione, Fe(II)SO₄, Cu(I) acetate, MgCl₂, ZnCl₂, CoCl₂,LiCl, acetonitrile, Trisma-Base, HCl, EDTA and H₂O₂ (30% v/v) were allpurchased from Sigma-Aldrich Inc. The Schlenk line was hand-made at theUniversity of Oslo and used with an in-house N₂ supply (99.999% pure).The N₂ gas was purchased from YARA PRAXAIR (Oslo, Norway).Oligosaccharides of N-Acetyl-D-glucosamine ranging from dimer to hexamerwere purchased from Seikagaku (Tokyo, Japan). Chitin beads for proteinpurification were purchased from New England Biolabs.

Nano-whiskers of beta-chitin were prepared according as described in(Fan et al., 2008, Biomacromolecules 9: 1919) by sonication of 3.0 mg/mLbeta-chitin particles suspended in 0.2 M acetic acid using a VibracellUltrasonic Processer equipped with a 3 mm sonication probe (Sonics,Newtown, Conn.) in four, one minute intervals with 30 s pauses betweeneach interval. Before use, the buffer in the chitin-whisker suspensionwas changed to 20 mM Tris pH 8.0 by dialysis. These whiskers were usedfor the experiment displayed in FIG. 2A, where an increased surface areawas needed to enable detection of CBP21 activity in the absence ofreductants and additional enzymes. All other experiments were performedwith non-treated beta-chitin.

Cloning, Expression and Purification of Recombinant Proteins

Chitin Binding Protein 21 (CBP21) from Serratia marcescens

CBP21 was cloned, produced and purified as previously described(Vaaje-Kolstad et al., 2005, J. Biol. Chem. 280: 11313). Briefly, the E.coli BL21 DE3 strain harbouring the pRSET-B vector containing the cbp21gene was grown overnight and harvested. The periplasmic content of thecells containing CBP21 was extracted by cold osmotic shock, filteredthrough a 0.2 micron syringe filter and kept at 4° C. Further, CBP21 waspurified from the periplasmic extract by chitin affinity chromatography,using chitin beads (NEB) as chromatographic medium. CBP21 was bound tothe column using 20 mM Tris pH 8.0 and 1.0 M (NH₄)₂SO₄ as bindingbuffer. After non-bound protein had passed through the column, thebinding buffer was run for one column volume before eluting CBP21 with20 mM acetic acid. The fraction containing CBP21 was concentrated usingan Amicon Ultra centrifugal filter unit with 10 kDa molecular weight cutoff, dialysed into 20 mM Tris-HCl pH 8.0 and stored at 4° C. until use.Protein purity was assessed by SDS-PAGE (always >99% pure) and proteinconcentration was determined by the Bio-Rad protein assay (Bio-RadLaboratories, Inc., USA) according to the instructions supplied by themanufacturer. Single site mutants of CBP21 were made as previouslydescribed (Vaaje-Kolstad et al., 2005, J. Biol. Chem. 280: 11313) usingthe QuickChange site-directed mutagenesis kit (Stratagene).

Chitinase C (ChiC) from Serratia marcescens

ChiC was cloned and produced as previously described (Synstad et al.,2008, Biosci. Biotechnol. Biochem. 72: 715). Briefly, E. coli BL21 DE3cells harbouring a pREST-B vector containing the chic gene under controlof the T7 promoter were grown to OD 0.6 and induced by 0.4 mM IPTG. Theenzyme was extracted from the cells by periplasmic extraction by coldosmotic shock. Further, ChiC was purified from the periplasmic extractby chitin affinity chromatography using chitin beads (NEB) as columnmaterial. Using 20 M Tris-HCl, pH 8.0 as running buffer, the periplasmicextract containing ChiC was passed through the column at 2.5 ml/minenabling binding of the chitinase to the chitin beads. After thenon-bound proteins in the extract had passed through the column, onecolumn volume of running buffer was run through the column beforeeluting the bound chitinase with 20 mM acetic acid. Following elution,the purified protein was concentrated using an Amicon ultrafiltrationdevice (Millipore) and finally dialysed into 20 mM Tris-HCl, pH 8.0 andstored at 4° C. before use. Protein purity was routinely assayed bySDS-PAGE and protein concentration was determined by the Bio-Rad method.

Chitin Deacetylase (AnCDA) from Aspergillus nidulans

Aspergillus nidulans FGSC A4 (obtained from FGSC) was grown at 37° C. insolid YAG medium (5 g/L yeast extract, 20 g/L glucose, 20 g/L agar, 1ml/L Cove's Trace Elements, 1.2 g/L MgSO₄.7H₂O) for 24-48 h to providean inoculum, and in liquid YG medium, typically for 16-24 hours withvigorous shaking (250-300 rpm). Genomic DNA was isolated with the SPFungi DNA Mini Kit (Omega Bio-tek, USA). The gene (GeneBank accessionnumber EAA66447.1) was amplified from Aspergillus nidulans FGSC A4genomic DNA using overlap extension polymerase chain reactions,excluding introns as well as the gene part coding for the signalpeptide. Primers for the reactions were P1f (Bgl/II): cga aqa tct acgcct ctg cct ttg gtt c (SEQ ID NO: 17), P2r: gag acg tgg tcg tat gta tgtgcg ccg act tga tg (SEQ ID NO: 18); P3f: caa gtc ggc gca cat aca tac gaccac gtc tcc ctc c (SEQ ID NO: 19); P4r: cca aca gtc gta get atc aac cctcga gca tta ac (SEQ ID NO: 20); and P5r (HindIII): cag aaq ctt tca atgata cca cgc aat ctc tcc atc acc gag aca atc acc aac agt cgt agc tat caac (SEQ ID NO: 21). A Bg/II and a HindIII site were incorporated at thestart and the end of the AnCDA gene to produce an in-frame N-terminalHis tag-fused construct in the pBAD/HisB(s) vector. This vector is avariant of the commercial vector pBAD/HisB (Invitrogen, USA) where theregion between the N-terminal polyhistidine tail and the multiplecloning site has been shortened (Kallio et al., 2006, J. Mol. Biol. 357:210). The resulting plasmids were transformed into Escherichia coliTOP10 cells (Invitrogen) and the inserted gene was sequenced at thesequencing facility of the Department of Chemistry, Biotechnology andFood Science at the Norwegian University of Life Sciences.

For protein expression, the transformed E. coli strain was grown at 37°C. in 2×TY medium (16 g/L Tryptone, 10 g/L yeast extract, 5 g/L NaCl)containing 100 mg of ampicillin per liter until OD₆₀₀=0.6 andsubsequently induced with 0.02% (w/v; final concentration) arabinosebefore further incubation at 28° C. overnight. Cells were harvested bycentrifugation and the protein was purified to homogeneity by Ni²⁺affinity chromatography. Protein concentrations were determined usingthe Bio-Rad protein assay, with bovine serum albumin as a standard.

E7 from Thermobifida fusca xy (Q47QG3)

The gene sequence encoding the mature variant of Q47QG3 from T. fusca xy(E7; residues 37-222) was cloned by amplifying the corresponding generegion from genomic DNA from T. fusca xy (purchased from ATCC) usingprimers designed according to the In-Fusion cloning protocol (Clontech).The resulting PCR product was inserted into a modified pRSETB vector(Invitrogen) using the In-Fusion™ technology (Clonetech) in-frame withthe signal peptide for direction of the protein product to the periplasmupon expression in E. coli. The modified pRSETB vector has the His-tagcontaining region replaced by the signal sequence encoding region of thecbp gene from Serratia marcescens (Vaaje-Kolstad et al., 2005, J. Biol.Chem. 280: 11313). By inserting genes of interest in frame with thesignal sequence, the gene product will be transported to the periplasmupon expression in E. coli and the exported protein will have a nativeN-terminus, meaning that the protein sequence starts with a histidine.Successful constructs were sequenced for verification and transformedinto E. coli BL21 DE(3) for protein expression. Cultures were grownovernight at 37° C. Cells were harvested by centrifugation and subjectedto periplasmic extraction by cold osmotic shock. The mature E7 proteinwas purified by chitin affinity chromatography using the chitin-beads tocapture the protein. The capture buffer contained 20 mM Tris-HCl pH 8.0and 1.0 M ammonium sulphate. The protein was eluted from the columnusing 20 mM acetic acid. Peaks containing pure protein were pooled andconcentrated using Sartorius Vivaspin devices with a 10 kDa cutoff.Using the same protein concentration device, the buffer was changed to20 mM Tris pH 8.0.

CelS2 from Streptomyces coelicolor A3(2) (Q9RJY2)

A gene encoding the mature form of Q9RJY2 from S. coelicolor A3(2)(CelS2; residues 35-364) was cloned by amplifying the corresponding generegion from genomic DNA from S. coelicolor A3(2) (purchased from ATCC)using primers designed according to the LIC cloning protocol (Novagen)that places a hexa-histidine tag art the N-terminus of the protein thatcan be removed using the Factor Xa protease, leaving no non-native aminoacids on the N-terminus of the protein. The PCR product was insertedinto the pET-32 LIC vector according to the instructions supplied by themanufacturer (Novagen). Successful constructs were sequenced forverification and transformed into E. coli Rosetta DE(3) for proteinexpression. Expression of soluble target protein was obtained by growinga 5 ml pre-culture of the transformed Rosetta DE(3) cells overnight at37° C., which was used the next day to innoculate a 300 ml volume ofLB-medium and growth was continued with shaking at 250 rpm at 37° C.Gene expression was induced by adding IPTG to a final concentration of0.1 mM when cell density reached an O.D. of 0.6, followed by immediatetransfer of the culture to a shaking incubator having 20° C. and forcontinuing the culturing overnight. The following day cells wereharvested by centrifugation. Cell pellets were resuspended in sonicationbuffer (20 mM Tris-HCl pH 8.0, 100 M PMSF, lysozyme and DNAse) followedby sonication using a Vibra Cell Ultrasonic processor equipped with a 3mm sonication probe (Sonics) in order to release cytoplasmic proteins.Cell debris was removed by centrifugation and His-tagged Q9RJY2 waspurified by standard IMAC (immobilized metal affinity chromatography)purification protocols using the Nickel-NTA IMAC resin (Qiagen).Purified protein was concentrated using Sartorius Vivaspin proteinconcentration devices with a 10 kDa cutoff, which also were usedconcomitantly to change to buffer into a buffer suitable for Factor Xaremoval of the His-tag (100 mM NaCl, 5.0 mM CaCl₂, 50 mM Tris pH 8.0).His-tags were cleaved off by adding Factor Xa and incubating overnightat room temperature, followed by His-tag removal using standard IMACchromatography. The flow through protein fraction containing theprocessed Q9RJY2 protein and Factor Xa was collected and concentratedusing Sartorius Vivaspin® protein concentration devices with a 10 kDacutoff. Finally, Factor Xa was removed using using Xarrest agarose beadsaccording to the manufacturer's instructions (Novagen). The buffer ofthe pure protein was changed to 20 mM Tris pH 8.0 using SartoriusVivaspin® protein concentration devices with a 10 kDa cutoff. Correctprocessing of the His-tag was verified by SDS-PAGE analysis.

Protein concentration was quantified using the Bio-Rad Bradford microassay (Bio-Rad) and protein purity was validated by SDS-PAGE.

Site-directed mutagenesis of the gene encoding the CelS2 protein wasdone using the Quickchange® Mutagenesis Kit (Stratagene) and accordingto instructions provided by the manufacturer. The mutated protein wasexpressed and purified using methods identical to those used for thewild-type protein.

Purification of Cel7A from Trichoderma reesei/Hypocrea jecorina

The endo-acting family GH7 cellulase Cel7A from Hypocrea jecorina waspurified from the commercially available H. jecorina extract CELLUCLAST™(Novozymes) using purification protocols described by Jager et al.,2010, Biotechnology for Biofuels 3(18). In short, the H. jecorinaextract was adjusted to 10 mM AmAc pH 5.0 and the enzyme was purifiedusing a DEAE-sepharose column attached to an Acta Purifier running 10 mMAmAc pH 5.0 as a mobile phase. Relevant fractions were pooled andconcentrated using Sartorius Vivaspin protein concentration devices witha 10 kDa cutoff. Purity was assessed using SDS-PAGE analysis.

Product Analysis by Mass Spectrometry (MS)

Matrix-Assisted Laser Desorption/Ionization—Time of Flight (MALDI-TOF)

Two microliter of a 9 mg/mL mixture of 2,5-dihydroxybenzoic acid (DHB)in 30% acetonitrile was applied to a MTP 384 target plate ground steelTF (Bruker Daltonics). One microliter sample was then mixed into the DHBdroplet and dried under a stream of air. The samples were analyzed withan Ultraflex MALDI-TOF/TOF instrument (Bruker Daltonics GmbH, Bremen,Germany) with a Nitrogen 337 nm laser beam. The instrument was operatedin positive acquisition mode and controlled by the FlexControl 3.3software package. All spectra were obtained using the reflectron modewith an acceleration voltage of 25 kV, a reflector voltage of 26, andpulsed ion extraction of 40 ns in the positive ion mode. The acquisitionrange used was from m/z 0 to 7000. The data were collected fromaveraging 400 laser shots, with the lowest laser energy necessary toobtain sufficient signal to noise ratios. Peak lists were generated fromthe MS spectra using Bruker FlexAnalysis software (Version 3.3).Post-source decay (PSD) spectra using the Bruker Daltonics LIFT systemwere recorded at 8 kV precursor ion acceleration voltage and fragmentacceleration (LIFT voltage 19 kV). The reflector voltage 1 and 2 wereset to 29 and 14.5 kV, respectively.

Product Analysis by HPLC and UHPLC

High Performance Liquid Chromatography (HPLC)

Isocratic HPLC was run on a Dionex Ultimate 3000 HPLC system set up witha 4.6×250 mm Amide-80 column (Tosoh Bioscience, Montgomeryville, Pa.,USA) with an Amide-80 guard column. The mobile phase consisted of 70%acetonitrile: 30% MilliQ H₂O and the flow rate was 0.7 ml/min. Elutedoligosaccharides were monitored by recording absorption at 190 nm.Chromatograms were recorded, integrated and analysed using theChromeleon 6.8 chromatography software (Dionex). The major product ofchitin degradation by ChiC is (GlcNAc)₂ (>95% of the total amount ofdegradation products on a molar basis), thus only (GlcNAc)₂ peaks weresubject for data analysis and used for quantification of the extent ofchitin degradation. A standard solution containing 0.10 mM (GlcNAc)₂ wasanalyzed at regular intervals during the sample series, and theresulting average values (displaying standard deviations of less than3%) were used for calibration.

For the sake of experimental simplicity and throughput, the degradationof chitin in reactions with ChiC and CBP21 was quantitatively assessedby measuring concentration of the dominant product, (GlcNAc)₂, only. Asa result of this simplification, product levels for maximally degradedchitin tend to be up to 25% lower than expected on the basis of thestarting concentration of chitin. This “loss” is due to the lack ofdetection of the following products: (1) longer oligomers and monomerswhich may amount to an estimated 5 wt. % of the total product mixture,(2) partially deacetylated products; the chitin that we use contains asmall fraction of deacetylated sugars (about 8%), (3) oxidized sugars.We have used conditions that boost CBP21 activity to a maximum. Theamounts of undetected oxidized sugars may amount to as much as 10-15% ofthe starting material (FIG. 16).

Ultra High Performance Liquid Chromatography (UHPLC)

UHPLC was run on an Agilent 1290 Infinity UHPLC system equipped withdiode array detector, set up with a Waters Acquity UPLC BEH amide column(2.1×150 mm with a 2.1×30 mm pre column both having a column materialparticle size of 1.7 μm) using 5 μL sample injections. Separation ofoxidized oligosaccharides was obtained at column temperature 30° C. anda flow of 0.4 mL/min starting at 72% ACN (A):28% 15 mM Tris-HCl pH 8.0(B) for 4 minutes, followed by an 11 minute gradient to 62% A: 38% Bwhich was held for three minutes. Column reconditioning was obtained bya two minute gradient to initial conditions and subsequent running atinitial conditions for 5 minutes. Eluted oligosaccharides were monitoredby recording absorption at 205 nm. Chromatograms were recorded,integrated and analysed using the ChemStation rev. B.04.02chromatography software (Agilent Technologies). The identity of theeluted oligosaccharides was verified by MALDI-TOF MS analysis accordingto the protocol described above.

Degradation Reactions and Sampling

General Reaction Conditions

Typical reactions were initiated by mixing beta-chitin (0.5 to 2 mg/mL)with CBP21 (0.1 to 1 μM), ChiC (0.5 μM) or AnCDA (1 □μM) or combinationsof these enzymes at a total volume of 0.5 ml in 1.5 ml plastic reactiontubes (Axygen Scientific Inc, Calif.) or in 1.8 ml borosilicate glassvials with screw cap tops and TEFLON® lined rubber septa. All reactionswere carried out in 20 mM Tris-HCl, pH 8.0 and incubated at 37° C. withshaking at 1000 rpm in an Eppendorf Thermo mixer unless statedotherwise. All reactions used for quantification were run intriplicates. All reactions used for qualitative purposes were repeatedat least three times.

Reactions with CBP21

Chitin solubilization by CBP21 was investigated by adding 1.0 μM CBP21to a reaction solution containing 2.0 mg/mL □beta-chitin and 5.0, 1.0 or0.2 mM ascorbic acid in 20 mM Tris-HCl pH 8.0. Reactions were incubatedat 37° C. and samples were taken at regular time intervals for analysisby MALDI-TOF MS and UHPLC. In order to investigate the effects ofreducing agents on the function of CBP21, ascorbic acid was exchangedwith either 1 mM reduced glutathione or 1 mM Fe(II)SO₄ in somereactions.

The effect of CBP21 on chitinase activity was studied by adding 0.5 μMChiC and 1.0 μM CBP21 to a reaction solution containing 2.0mg/mL□beta-chitin and 1.0 mM ascorbic acid in 20 mM Tris-HCl pH 8.0. Thereaction was incubated at 37° C. and sampled at regular time intervals.Chitin degradation was measured by determining the concentration of(GlcNAc)₂ by HPLC. Control experiments where CBP21 and/or ascorbic acidwere excluded from the reaction solution were performed in the samemanner.

To investigate whether CBP21 was capable of cleaving and/or oxidizingsoluble substrates, a 500 μL reaction solution containing 1.0 μM CBP21,100 μM (GlcNAc)₆ (0.12 mg/mL) and 1.0 mM ascorbic acid all dissolved in20 mM Tris pH 8.0 was incubated for 16 hours at 37° C. before productanalysis by MALDI-TOF MS. The same was performed for control reactionswhere either CBP21 or ascorbic acid or both were excluded from thereaction solution. An experiment designed to visualize the range ofpolymeric products generated by CBP21 was performed by combining 1.0 μMCBP21 and 1.0 μM□AnCDA in a reaction solution containing 2.0mg/mL□beta-chitin, 1.0 mM ascorbic acid and 10 μl CoCl₂ (necessary forfull AnCDA activity) in 20 mM Tris-HCl pH 8.0. Control reactions wereperformed where CBP21 was excluded from the reaction solution and/orreplaced by 0.5 μM ChiC. The reactions were incubated for 16 hours at37° C., followed by product analysis with MALDI-TOF MS.

Molecular Oxygen Free Reaction and Related Control Reactions

In order to obtain a di-oxygen free reaction solution, all reactioncomponents except the enzyme or enzymes were mixed in a glass vialclosed with a screw cap containing a rubber septum and degassed using aSchlenk line. The enzyme was added to a separate vial which was treatedidentically to the vial containing the reaction mixture. Before startingthe degassing procedure, a freshly made 1.0 M dithionite solution wasadded the reaction solution to yield a final concentration of 10 mM toensure total removal of molecular oxygen in the solution. The degassingprocedure was performed by penetrating the rubber septum of the sealedvial with a needle connected to the Schlenk line, followed by fivecycles of 5 minute degassing (vacuum) and 1 minute of N₂ saturation. Thefinal cycle left the vials slightly pressurized by N₂. After degassingboth the reaction solution and the enzyme solution, a syringe was usedto withdraw an appropriate amount of the enzyme solution which then waspromptly injected into the vial containing the reaction solution inorder to initiate the reaction, while injecting bubbles of air wasavoided. The effect of a molecular oxygen free environment was assessedby analyzing the activity of 1.0 μM CBP21 on 2.0 mg/mL beta-chitin inthe presence of 1.0 mM ascorbic acid in 20 mM Tris-HCl pH 8.0 byMALDI-TOF MS. Additionally, the degradation of 0.1 mg/ml beta-chitin by0.5 μM ChiC in the presence of 1.0 μM CBP21 and 1.0 mM ascorbic acid in20 mM Tris-HCl pH 8.0 was analyzed in the same di-oxygen freeenvironment. Samples were analysed by HPLC after 16 hours incubation at37° C.

This latter experiment was also conducted in the absence of sodiumdithionite and at a higher chitin concentration (0.45 mg/mL), but withotherwise identical reaction conditions. It should be noted that eventhough every precaution was taken to avoid oxygen entering the reactionsolution, the dioxygen removal is not 100% efficient. This can be seenby studying the result from the ¹⁸O₂ experiment (see FIG. 7C), whereproducts resulting from oxidation by ¹⁶O₂ are detected.

Additional control experiments were performed by running experimentswhere 0.45 mg/mL beta-chitin, 1.0 μM CBP21, 0.5 μM ChiC and 1.0 mMreduced glutathione in 20 mM Tris-HCl pH 8.0, were run in the presenceof either 2.0 mM sodium azide or 2.0 mM potassium cyanide. The reactionswere incubated at 37° C., sampled at 30, 60 and 90 minutes, and productswere analyzed by UHPLC.

Reactions Under Metal Chelating Conditions

Divalent cations were removed by chelation through dialysis of a 10mg/mL CBP21 solution in a buffer containing 20 mM Tris-HCl and 5 mMEDTA. The protein solution was present in a Slide-A-Lyzer cassette(Pierce) with 10 kDa MW cut-off dialysis membrane. Dialysis wasperformed for 16 hours at 4° C. with a protein to buffer volume ratio of1:1000 with moderate magnetic stirring. Reactions with metal-free CBP21were performed as described above, except that EDTA was added to thereaction buffer to a final concentration of 5 mM. Re-activation ofmetal-free CBP21 was achieved by adding either ZnCl₂ or MgCl₂ to thereaction mixture to a final concentration of 25 mM. Reactions were runfor 180 minutes and sampled at 30 minute intervals. For there-activation experiment, the divalent cations were added to theappropriate reaction solutions immediately after the third sampling (90minutes). Chitin degradation was measured by determining theconcentration of (GlcNAc)₂ by HPLC.

Reactions in Buffered H₂ ¹⁸O

Beta-chitin and ascorbic acid were each suspended/dissolved in H₂ ¹⁸O toyield concentrations of 2 mg/mL and 1.0 M, respectively. In order toachieve the correct pH in the H₂ ¹⁸O reaction solution 10 μL 1.0 MTris-HCl pH 8.0 was transferred to a glass vial, which was heated withdry air at 60° C. until all liquid had evaporated. 498 μL of thebeta-chitin suspension was transferred to the same glass vial achievingthe intended pH for the reaction. Concomitantly 0.5 μL ascorbic acid(dissolved in H₂ ¹⁸O) and 0.75 μL of a 660 μM solution of CBP21(dissolved in H₂ ¹⁶O) were added to the solution to start the reaction,yielding final concentrations of 1 mM for ascorbic acid and 1 μM forCBP21. The glass vial was sealed with a screw cap with a TEFLON® coatedrubber liner to ensure as little as possible contamination of H₂ ¹⁶Ofrom the air phase into the reaction solution. After incubation for 16hours at 37° C. reaction products were analyzed by MALDI-TOF MS.

Reactions in an ¹⁸O₂ Gas Saturation Solution

In a glass vial containing a reaction mixture of 2.0 mg/mL beta-chitinand 1.0 mM ascorbic acid in 20 mM Tris pH 8.0, CBP21 was added to yielda final concentration of 1.0 μM. Immediately after reaction initiation ascrew cap containing a TEFLON® lined rubber septum was used to close thevial and the Schlenk line was used to remove dissolved molecular oxygenand fill the head space with N₂ (according to the procedure describedunder the heading “molecular oxygen free reaction”). After the fivecycles of degassing and N₂ filling were completed, a gas cylindercontaining compressed ¹⁸O₂ gas was connected to the vial by pushing aneedle through the septum of the vial. The vial was then placed undervacuum, removing atmospheric gas residing in the tubing and the headspace of the vial. After isolating the vial and the ¹⁸O₂ gas cylinderfrom the rest of the Schlenk line by closing appropriate in-line valves,the head space of the vial was filled with ¹⁸O₂ gas by slowly openingthe gas cylinder regulator. The vial was then removed from the needleconnections and after incubation at 37° C. for 16 hours reactionproducts were analyzed by MALDI-TOF MS.

Reaction of Beta-Chitin with Fenton Chemistry

In order to determine whether Fenton chemistry (Fe²⁺ and H₂O₂ combinedin an oxygen saturated solution to yield reactive hydroxyl radicals;Sawyer et al., 1996, Acc. Chem. Res. 29: 409) would yield solubleproducts from chitin, 2 mg/mL beta-chitin suspended in 20 mM Tris-HCl pH8.0 was incubated for 16 hours with 10 mM Fe(II)SO₄ and 0.3, 0.03 or0.003% (v/v) H₂O₂ in plastic sample tubes having perforated lids (forrelease of gas generated during the reaction). Samples were analyzed byMALDI-TOF MS.

Control Experiment With Another CBM33 Protein Identified by GenomeMining—CBM33 (EF0362) from Enterococcus feacalis

The gene encoding the mature family 33 CBM from Enterococcus faecalis,EfCBM33 (Uniprot ID:Q838S1; EF0362; uniprot.org/uniprot/Q838S1), withoutits native leader peptide, was cloned into the pRSET-B-CBP21 vector inframe with the CBP21 leader peptide, replacing the gene encoding CBP21.The protein was expressed in E. coli BL21 DE3 cells, harvested from theperiplasmic fraction by cold osmotic shock and purified to homogeneityby chitin affinity chromatography. Thus, this protein was expressed andpurified in exactly the same way as CBP21, using CBP21's leader peptideto drive secretion. The fractions containing pure protein (assessed bySDS-PAGE) were pooled and concentrated using an Amicon centrifugalconcentrator with 10 kDa cutoff to yield a 20 mg/ml solution. Theprotein was crystallized by hanging drop vapor diffusion experimentsusing a crystallization liquor containing 1.0 MK/Na Tartrate, 0.1 Mimidazol pH 8.0 and 0.2 M NaCl. Crystals pyramidal in shape andmeasuring approximately 0.2 mm in width (see FIG. 15A) were obtainedafter 48 hours incubation at room temperature. A 0.95 Å dataset has beencollected from a single crystal and the structure has been solved bymolecular replacement using CBP21 (PDB ID 2BEM) as template. Refinementis complete, but not published. The quality of the data is illustratedby FIG. 15B. FIG. 15C shows a structural superposition of CBP21 andEfCBM33.

For use in chitin degradation experiments, eight crystals were harvestedfrom a 2 μL drop using a nylon loop and transferred to a 4 μL dropcontaining the crystallization liquor from the buffer reservoir. Thecrystals were mixed around in order to “rinse off” potentialcontaminants. After the first rinse, the crystals were transferred to anew 4 μL drop containing the crystallization liquor for a second rinsingcycle. Finally, all crystals were transferred to and dissolved in a 4 μLdrop containing 20 mM Tris-HCl, pH 8.0. The resulting solution wasdiluted by adding it to a test tube containing 46 μL 20 mM Tris-HCl, pH8.0. For reactions with beta-chitin, 5 μL of the EfCBM33 solution wasmixed with a 95 μL solution containing 2 mg/ml beta-chitin and 2 mMascorbic acid in 20 mM Tris-HCl pH 8.0; the reaction mixture was thenincubated for 90 minutes at 37° C. in a test tube incubator rotating at1400 rpm. Soluble products in the supernatant of the reaction wereanalyzed by MALDI-TOF, using the same methods as those used for testingthe activity of CBP21. Further, the ability of EfCBM33 to boostdegradation of alpha-chitin was probed by conducting an experiment where2.0 mg/ml alpha-chitin (shrimp shells) was incubated with 0.3 μM of thechitinase from Entreococcus feacalis (protein name (EF0361)) in thepresence or absence of 0.3 μM EfCBM33 and 1.0 mM reductant (R: reducedglutathione) incubated at 37° C. with agitation at 900 rpm. A boost ofthe chitinase activity is clearly observed in the presence of EfCBM33and reductant.

Determination of CBP21 Reaction Speed and Degree of Substrate Oxidiation

Using the UHPLC method for separating oxidized chitooligosaccharides,pure GlcNAc3GlcNAcA and GlcNAc4GlcNAcA samples were obtained byfractionation of beta-chitin samples treated by CBP21 in the presence ofascorbic acid. Fractions were dried under vaccum (SpeedyVac), andresuspended in 50 μL MilliQ water. Purity was verified by MALDI-TOF MS.Isolated GlcNAc3GlcNAcA or GlcNAc4GlcNAcA were each incubated for 2hours at 37° C. with 7.0 μM of a pure recombinant family 19 chitinase(ChiG from Streptomyces coelicolor (Hoell et al., 2006, FEBS J. 273:4889)) resulting in production of equimolar amounts of GlcNAc2 andGlcNAcGlcNAcA or GlcNAc2GlcNAcA, respectively. The amount of GlcNAc2resulting from the hydrolysis was estimated using a predeterminedstandard curve. Response factors for the GlcNAcA containingoligosaccharides were obtained by determining GlcNAcGlcNAcA/GlcNAc2 andGlcNAc2GlcNAcA/GlcNAc2 peak area ratios and found to be 0.71 and 0.81,respectively. A response factor for GlcNAc3GlcNAcA was approximated tobe 0.88 by extrapolation of the two experimentally determined responsefactors. Using the response factors determined, GlcNAcGlcNAcA andGlcNAc2GlcNAcA peaks could be quantified using GlcNAc2 for calibration.In experiments for the simultaneous detection and quantification ofGlcNAc2 and oxidized oligomers 1.0 mM of reduced gluthathione was usedas reductant instead of ascorbic acid because the latter interferes withthe chromatographic analysis. Additionally, the reactions contained 0.45mg/mL beta-chitin, 1.0 μM CBP21 and 0.5 ChiC μM in 20 mM Tris-HCl pH8.0.

The reactions were incubated at 37° C. in an Eppendorf Thermo mixer withshaking at 1000 rpm, and sampled at 30, 60, 120 and 300 minutes. Allsamples were mixed 1:1 with 100% acetonitrile in order to stop thereaction and soluble products were analyzed by UHPLC. Separation of theoxidized oligosaccharides and GlcNAc2 was achieved using a columntemperature of 30° C. and a flow of 0.4 mL/min, with a gradient startingat 80% ACN (A):20% 15 mM Tris-HCl pH 8.0 (B) for 4.5 minutes, followedby an 11 minute gradient to 63% A: 37% B which was held for 3.5 minutes.Column reconditioning was achieved by a two minute gradient to initialconditions and subsequent running at initial conditions for 5 minutes.Eluted oligosaccharides were monitored by recording absorption at 205nm. Chromatograms were recorded, integrated and analysed using theChemStation rev. B.04.02 chromatography software (Agilent Technologies).

In order to approximate the rate of the CBP21 oxidohydrolytic activity,reactions containing 0.45 mg/mL beta-chitin, 1.0 μM CBP21 and 1.0 mMreduced glutathione in 20 mM Tris-HCl pH 8.0 were incubated at 37° C.and sampled at 10, 15, 30,45, 60 and 300 minutes. Instead of stoppingthe reaction with acetonitrile, a cocktail of purified recombinantchitinases containing 28 μM ChiC (see above), 71 μM ChiG (Hoell et al.,2006, supra), 63 μM ChiB (Brurberg et al., 1995, Microbiology 141: 123,Brurberg et al., 1996, Microbiology 142: 1581) and 15 μM ChiA (Brurberget al., 1996, supra, Brurberg et al., 1994, FEMS Microbiol. Lett. 124:399) was added to the sample (0.1 volume) in order to obtain rapidcomplete degradation of the chitin. Under these conditions, insolublechitin completely disappeared within 30 minutes. The quantities of theoxidized products (exclusively GlcnAcGlcNAcA and GlcNAc2GlcNAcA) weredetermined using the UHPLC method outlined above.

Analysis and Quantization of Glucose and Cellobiose by HPLC (E7 andCelS2)

Samples containing glucose and cellobiose were analysed by isocraticHPLC run on a Dionex Ultimate 3000 HPLC system set up with a 7.8×100 mmRezex RFQ-Fast Fruit H+ column (Phenomonex) heated to 80° C. The mobilephase consisted of 5 mM sulfuric acid and the flow rate used was 1.0ml/min. Eluted glucose and cellobiose were monitored by recordingrefractive index. Quantification was obtained by running glucose andcellobiose standards. Chromatograms were recorded, integrated andanalysed using the Chromeleon 6.8 chromatography software (Dionex).

Analysis of Native and Oxidized Cellooligosaccharides Using HPAEC (E7and CelS2)

Separation of native and oxidized cellooligosaccharides was achievedusing a Dionex Bio-LC equipped with a CarboPack PA1, a columntemperature of 30° C. and a flow of 0.25 ml/min, with startingconditions, i.e., 0.1 M NaOH. A stepwise linear gradient with increasingamounts of sodium acetate was applied, going from 0.1 M NaOH and 0.1 Msodium acetate in 10 minutes, then to 0.1 M NaOH and 0.3 M sodiumacetate for 25 minutes then increasing to 0.1 M NaOH and 1.0 M sodiumacetate at 30 minutes which was kept for 10 minutes. Columnreconditioning was achieved by a one minute gradient to initialconditions and subsequent running at initial conditions for 14 minutes.Eluted oligosaccharides were monitored by PAD detection. Chromatogramswere recorded and analysed using Chromeleon 7.0 Peak identification wasachieved by a procedure including the following steps: oxidizedcellooligosaccharides were separated using a Dionex Ultimate 3000 UHPLCsystem carrying a Hypercarb 150×2.1 mm column (Thermo Scientific)running a gradient of water/0.1% TFA and acetonitrile/0.1% TFA (from 20to 80% acetonitrile) according to the method developed by Westphal etal., 2010, Journal of Chromatography A 1217: 689-695. Eluted peaks weremanually fractioned, freeze-dried, re-dissolved in MilliQ water andidentified using MALDI-TOF MS. Pure oxidized cellooligosaccharides withknown identity were then analyzed using the HPEAC method described abovein order to establish the identity of the oxidized cellooligosaccharidesgenerated by CelS2 and E7.

Cellulose Degradation Experiments (E7 and CelS2)

Assays performed to evaluate the function of E7 and CelS2 were set upusing a variety of cellulosic substrates (AVICEL®, filter paper andsteam exploded wood chips from poplar), at either pH 5.5 (20 mM sodiumacetate buffer), 6.5 (20 mM Bis-Tris buffer) or 8.0 (20 mM Tris buffer)in reaction mixtures containing 1.0 mM MgCl₂. The effect of the presenceof an external electro donor was probed by adding 1.0 mM or 0.5 mMreduced glutathione or ascorbic acid to the reaction mixture (see figurelegends for details).

Results

We show here that CBP21, a single-domain protein comprising one CBM33domain, in fact is an enzyme that catalyzes an oxidohydrolytic cleavageof glycosidic bonds in crystalline chitin, thus opening up theinaccessible polysaccharide material for hydrolysis by normal glycosidehydrolases. This enzymatic activity was first discovered when wedetected traces of non-native chito-oligosaccharides upon incubation ofbeta-chitin nano-whiskers with CBP21 (FIG. 2A). The products wereidentified as chitin oligosaccharides with a2-(acetylamino)-2-deoxy-D-gluconic acid (GlcNAcA) at the reducing end(FIGS. 2B, 3A, 3B, 3C, and 3D). We then found that addition ofreductants dramatically increased the efficiency of the reaction (FIGS.4, 6A, and 6B), enabling the destructuring of large crystallinebeta-chitin particles by CBP21 alone (FIG. 2C), releasing a range ofoxidized products (FIGS. 5A and 5B and below) and boosting chitinaseefficiency to much higher levels than previously observed (compare FIG.10 with FIG. 4).

If CBP21 acted randomly on crystalline surfaces, one would expectgeneration of longer oligosaccharides, which are difficult to detect dueto their low solubility. The majority of soluble products generated byCBP21 in the presence of a reductant had a DP below 10 (FIG. 2A; FIGS.5A and 5B). To visualize longer products, we exploited a newly clonedchitin deacetylase from Aspergillus nidulans (AnCDA) to increase thesolubility of longer chitin fragments by deacetylation. This approachindeed revealed the formation of chitin fragments with high DP, eitherwith CBP21 (FIG. 2D) or with an endochitinase (ChiC; FIG. 2E). BothCBP21 and ChiC generated long products, indicative of an “endo-” type ofactivity.

Two important features stand out. Firstly, when using CBP21 all detectedproducts are oxidized (i.e., they contain a GlcNAcA moiety), confirmingthe observation that CBP21 catalyzes oxidative hydrolysis of glycosidicbonds. Secondly, whereas the products released by ChiC represent acontinuum of lengths, the products released by CBP21 are dominated byeven-numbered oligosaccharides (FIGS. 2D, 6A, and 6B). This shows thatChiC tends to cleave any glycosidic bond, whereas CBP21 shows a strongpreference for cleaving every second glycosidic bond. Keeping in mindthe disaccharide periodicity in the substrate (FIG. 1A), this crucialobservation implies that ChiC approaches single polymer chains from “anyside”, whereas CBP21 must approach the substrate from one fixed side.The latter situation would be obtained if the polysaccharide chaincleaved by CBP21 is part of an intact crystalline structure. Clearly,the endochitinase ChiC and CBP21 have different roles in chitinhydrolysis.

The CBP21 mediated cleavage mechanism was probed in more detail byisotope-labelling. Experiments in H₂ ¹⁸O showed that one of the oxygenatoms introduced at the oxidized new chain end comes from water (FIG.7A). The only plausible source for the second oxygen was molecularoxygen and this was confirmed by experiments performed in ¹⁸O₂saturating conditions (FIGS. 7B and 7C). Removal of dissolved molecularoxygen or in the reaction solution inhibited CBP21 activity (FIGS. 8A,8B, and 8C), confirming the requirement for molecular oxygen forcatalysis. The strong inhibition by cyanide, a common mimic of molecularoxygen, supports the crucial role of the oxidative step (FIGS. 8C and8D). Thus, the reaction catalyzed by CBP21 comprises a hydrolytic stepand an oxidation step, as summarized in FIG. 7D. Enzymaticoxidohydrolysis of polysaccharides has so far not been described. CBP21is referred to herein as a “chitin oxidohydrolase”. Likewise, GH61proteins are referred to as “cellulose oxidohydolases”.

CBP21 catalysis was found to be dependent on the presence of a divalentcation (FIG. 9), which may bind to the conserved histidine motif (FIG.1F). Interestingly, the activity of GH61 proteins also depends ondivalent cations (Harris et al., 2010, Biochemistry 49: 3305).Structural studies of both CBP21 (Vaaje-Kolstad et al., 2005, J. Biol.Chem. 280: 11313) and GH61 proteins (Harris et al., 2010, Biochemistry49: 3305; Karkehabadi et al., 2008, J. Mol. Biol. 383: 144) showconsiderable structural plasticity in the metal-binding site, explainingwhy the metal binding site is promiscuous and why the need for divalentcations is rather unspecific (as shown in FIG. 9 and in Harris et al.,2010, Biochemistry 49: 3305). Mutation of the second histidine (His114in CBP21 and His89 in GH61E from Thielavia terrestris) in the metalbinding motif knocked out activity of both proteins (FIGS. 10A and 10Band Harris et al., 2010, Biochemistry 49, 3305, respectively). It isconceivable that the metal ion binding aids directly in thebinding/stabilization/activation of the reactive oxygen species and/orthe hydrolytic water during catalysis. Alternatively, the metal ion maybe essential for stabilizing the structure of the active site region ofthe protein without interacting directly with the oxygen or the water.

Reductants boosted the oxidohydrolytic activity of CBP21 to extremelevels (FIGS. 2C, 4, 6A, 6B, 11A, and 11B), which very well may bebeyond what is ever achieved in nature. The reductants are likely tofunction as electron donor in the oxidative enzymatic process, but theexact flow of electrons from the reductant to molecular oxygen is notyet clear. One option is that electron donation takes place via thegeneration of reactive oxygen species such as O₂ ⁻. Another option isthat electrons are somehow channelled to a complex involving CBP21, thesubstrate and O₂, implying that reactive oxygen species such as O₂ ⁻only emerge on the substrate and will not or hardly be present insolution. Most interestingly, and of biotechnological importance, ourdata show that activity of these new enzymes can be boosted considerablyby simply adjusting the reaction conditions.

We conducted numerous control experiments that all confirmed theconclusion that formation of oxidized products only occurs in thepresence of CBP21 and crystalline substrates. The presence of reductantsalone did not yield oxidized products (FIG. 12) and did not potentiateor inhibit chitinase action (FIGS. 4 and 10B). When incubated underoptimal conditions with hexameric N-acetylglucosamine, neitherdegradation products nor oxidized oligosaccharides were observed FIGS.13A, 13B, 13C, and 13D). We also studied the possibility that CBP21would work without directly interacting with the cleaved bond itself,since one could envisage some sort of CBP21-induced “destructuring”mechanism making the substrate more accessible for the action ofreactive oxygen species generated in a CBP21-independent manner, e.g.,by Fenton chemistry. We could however not detect any soluble productsupon subjecting beta-chitin to Fenton chemistry (FIG. 14). All theseexperiments confirm that CBP21 actively participates in theoxidohydrolytic cleavage reaction.

As shown in FIGS. 8A, 8B, 8C, and 8D, CBP21 activity is stronglyinhibited by cyanide, a known O₂ mimic, but not by azide, a knowninhibitor of haem proteins. Several reductants capable to function aselectron donors boosted the activity of CBP21 (FIGS. 2C, 11A, and 11B).The experimental data indicate that the oxidation step catalyzed byCBP21 is co-factor independent and depends on an external electrondonor. Co-factor independent oxygenases have been described before, butthese enzymes are normally thought to use conjugated carbanions in thesubstrates as electron donors (Fetzner and Steiner, 2010, Appl.Microbiol. Biotechnol. 86:791), a mechanism that is not likely in thecase of a polysaccharide substrate. If the oxidation step was to happenfirst, this would imply that CBP21 catalyzes co-factor independentoxygenation of a saturated carbon, which is unprecedented and perhapsnot very likely. On the other hand, such a mechanism could yield anintermediate product, for example an ester bond, that may be more proneto hydrolysis than the original glycosidic bond. Alternatively, thehydrolytic step could occur first, which would imply that CBP21 iscapable of hydrolyzing glycosidic bonds in a crystalline environmentusing a hitherto unknown mechanism. Such a hydrolytic step would requiresome degree of substrate distortion (Davies et al., 2003, Mapping theconformational itinerary of beta-glycosidases by X-ray crystallography.Biochem. Soc. Trans. 31: 523 and Vocadlo and Davies, 2008, Mechanisticinsights into glycosidase chemistry. Curr. Opin. Chem. Biol. 12: 539),which seems challenging in a crystalline packing. However, in favour ofthis mechanism, the subsequent oxidation of the resulting sugar aldehyde(“reducing end”) is more straightforward than oxidation of a saturatedcarbon.

CBP21 introduces chain breaks in what probably are the most inaccessibleand rigid parts of crystalline polysaccharides substrates and its modeof action differs fundamentally from the mode of action of glycosidehydrolases. The key difference is that the glycoside hydrolases aredesigned to host a single “soluble” polysaccharide chain in theircatalytic clefts or pockets and that their affinity and proximity to thecrystalline substrate tends to be mediated by non-hydrolytic bindingdomains. In contrast, CBP21 and GH61 enzymes have flat surfaces (FIGS.1D and 1E) that bind to the flat, solid, well-ordered surfaces ofcrystalline material and catalyze chain breaks by an oxidohydrolyticmechanism. The chain break will result in disruption of crystallinepacking and increased substrate accessibility, an effect that may beaugmented by the modification of one of the new chain ends. At thecleavage point one of the new ends is a normal non-reducing end(indicated by R—OH in FIG. 7D). The other new end would have been a newreducing end if the cleavage had been performed by a normal glycosidehydolase. However, in this case the product is different and the lastsugar is oxidized to become 2-(acetylamino)-2-deoxy-D-gluconic acid(FIGS. 2B and 7D; GlcNAcA). This new “acidic chain end” will interferewith normal crystal packing because it will not have the normal chairconformation of the sugar ring and because it carries a charge.

The enzyme activity demonstrated in this study is difficult to discover,because one depends on detecting products with low solubility andpotentially a high tendency to remain attached to the crystallinematerial. In this sense, working with chitin is easier than working withcellulose because product solubilities are slightly higher and becausecrystalline packing is less compact (Eijsink et al., 2008, TrendsBiotechnol. 26: 228). The experiment with the chitin deacetylase (FIGS.2D and 2E) provided essential insights, but this approach can simply notbe used for cellulose. Looking for chain-breaking activities of GH61proteins with commonly used reducing end assays obviously will not workdue to the oxidative mechanism.

Using methods for quantification of oxidized products, we were able toestimate the speed and degree of oxidation under various conditions(FIGS. 16A and 16B). When CBP21 acts alone on beta-chitin under optimalconditions the oxidation rate is in the order of 1 per minute and themaximum extent of oxidation is about 7.6% of the sugars. Simultaneousdegradation of beta-chitin with CBP21 and ChiC under optimal conditionsled to oxidation of approximately 4.9% of the sugars. It must be notedthat in nature enzymes such as CBP21 normally act simultaneously with atleast one, and in the case of S. marcescens, three chitinases (Suzuki etal., 1998, Biosci. Biotechnol. Biochem. 62: 128).

The experiments conducted on CBM33 family protein EfCBM33 revealed thatthis protein is functionally similar to CBP21. The results of the MSanalysis, depicted in FIG. 15D resemble those obtained with CBP21,clearly showing the formation of oxidized products and theoxidohydrolytic properties of this protein. It is interesting to notethat EfCBM33 not only works on beta-chitin (FIG. 15D but also onalpha-chitin (FIG. 15E).

The efficacy of CBM33 proteins on cellulose as the substrate wereexamined. Mature wild type E7, CelS2 and a cellulase classified as abelonging to GH family 7 and originating from Trichoderma reesei calledCel7A (Harjunpaa et al., 1999, FEBS Letters 443: 149-153) were purifiedto ˜95% purity using the cloning, expression and purification strategiesdescribed in the Material & Methods section. Finally, a cellulasemixture called CELLUCLAST™, which is an easily available and well knowncommercial product from Novozymes, was used.

To determine whether CelS2 and/or E7 had the ability to release solublesugars from crystalline cellulose, CelS2 or E7 was incubated withAVICEL® in the presence or absence of an external electron donor(reduced glutathione or ascorbic acid). Putative reaction products wereanalyzed using MALDI-TOF MS for qualitative detection of product typesand HPAEC (high pressure anion exchange chromatography), achromatographic method enabling product identification and, inprinciple, quantification. Indeed, soluble oligomeric products wereobserved with both proteins (only shown for CelS2; FIGS. 17-19) and withboth detection methods. Native cellooligosaccharides were observed inboth conditions with and without external electron donor, but insubstantial larger amounts in the former condition. Oxidizedcellooligosaccharides were only observed in experiments where anexternal electron donor was present.

More specifically, MALDI-TOF MS analysis revealed the presence ofcellooligosaccharides with an oxidized reducing end (i.e.,cellooligosaccharides with the reducing glucose moiety replaced with agluconic acid moiety; FIGS. 17 and 18). The degree of polymerization(DP) of the released soluble oxidized oligosaccharides visible in FIG.17 ranged from DP4 to DP7 and the product signals showed alternatingintensity/quantity depending on the cellooligosaccharide being even orodd numbered in terms of DP (even numbered cellooligosaccharidesdominated). HPAEC analysis of the same samples reflected the resultsprovided by the MALDI-TOF MS analysis, showing the presence of peakswith alternating intensities representing oxidized cellooligosaccharides(FIG. 19).

The dominance of even numbered oligosaccharides is a logical consequenceof the fact that CBM33 enzymes attack the polysaccharide chains in theircrystalline environment. As the repeating unit of cellulose and chitinis a dimer, only every second sugar/glycosidic bond on the crystallinepolysaccharide chain is prone for cleavage by the CBM33 enzyme, meaningthat released products would tend to have an even-numbered DP. (NB. acidhydrolysis of either crystalline cellulose or chitin gives an evendistribution of even and odd numbered oligosaccharides).

FIG. 19 shows that incubation with CelS2 in the presence of a reducingagent, leads to cleavage of the cellulose chains in the crystal in anoxidohydrolytic manner, releasing soluble oxidized cellooligosaccharides(light grey, upper line). This also leads to additional release ofnon-oxidized cellooligosaccharides, which is likely to result in partfrom the disruption the AVICEL® crystal surface that facilitates therelease of shorter oligomers that are captured in the crystal.Additionally, chain cleavage by CelS2 near the reducing end of acellulose chain will give rise to such products.

Reactions containing crystalline cellulose (filter paper) and cellulases(either a cellulase mixture called CELLUCLAST™, or a single componentcellulase, Cel7A) were monitored for cellobiose and glucose release inthe presence and absence of CelS2/E7 and/or an external electron donor.

When CELLUCLAST™ was incubated with filter paper in the presence ofCelS2 or E7, the glucose yield was indeed higher than when the filterpaper was incubated with only CELLUCLAST™ (FIG. 20; ˜3.0 fold increasefor CelS2 and E7 after 24 hours incubation and ˜9 fold increase forCelS2 and ˜3.5 fold increase for E7 after 120 hours incubation). Whenboth CelS2 and an external electron donor were added, the glucose yieldswere even higher (FIG. 20; ˜5 fold increase for CelS2 and 3.5-foldincrease for E7 after 24 hours incubation and ˜11 fold increase forCelS2 and ˜4.5 fold increase for E7 after 120 hours incubation),indicating that the presence of an external electron donor indeed is ofimportance for the boosting effect of CelS2 and E7.

Since the CELLUCLAST™ product contains a complex mixture of hydrolyticactivities that may complicate the interpretation of the results, amonocomponent cellulase (Cel7A) was purified from CELLUCLAST™(Novozymes) and used to probe the boosting efficiency of CelS2. Effectssimilar to those observed when combining CELLUCLAST™ and CelS2 in thepresence and absence of an external electron donor were also observedwhen incubating Cel7A with CelS2 (FIG. 21). In the absence of anexternal electron donor, CelS2 improved Cel7A activity (in terms ofcellobiose yield) 5.5- and 7.3-fold after 18 and 40 hours incubation,respectively. In the presence of an external electron donor, CelS2improved Cel7A activity 6.7- and 9.8-fold after 18 and 40 hoursincubation, respectively.

FIGS. 20 and 21 show that in addition to boosting cellulose activity thepresence of CelS2 in both cases also aids in maintaining the cellulaseactivity over time. Whereas the activity of the cellulases incubated inthe presence or absence of an external electron donor comes to an almostcomplete stop within the first 18-24 hours, cellulase activity continuesin the samples incubated with CelS2. This effect may be a partialexplanation for the synergistic effects that CBM33 proteins have on thedegradation of chitin or cellulose by hydrolytic enzymes (chitinase andcellulases, respectively).

The oxidized products generated by CelS2 in the presence of an externalelectron donor exists in an equilibrium of two forms, the gluconodelta-lactone which gains in population at mildly acidic pH and thegluconic acid form that dominates at mildly alkaline pH. At mildlyalkaline pH (e.g., pH 8.0) it is likely that the charge developing onthe cellulose crystal surface due to CelS2 activity may aid thedistortion and disruption/solubilization of the cellulose crystal andthus increase the accessibility of the substrate for the cellulases.However, it is conceivable that oxidized oligosaccharides and oroxidized chain ends in cellulose crystals may also inhibit certaincellulases (e.g., exo-acting enzymes), making it likely that the degreeof CelS2/CBM33 activity should be carefully adjusted for optimalboosting efficiency.

When probing the cellulose boosting properties of CelS2 at various pHsusing monocomponent Cel7A as the cellulose hydrolytic component, pH 5.5came out as the optimal pH for activity. Less activity was seen at pH6.5 and no activity could be detected at pH 8.0 (data not shown). Themost obvious reason for the decrease in cellulose hydrolysis atincreasing pH is the pH stability and efficiency of Cel7A. The cellulaseis close to inactive at pH 8.0, which is a common trait for fungalcellulases in general (Garg & Neelakantan, 1981, Biotechnology andBioengineering 23: 1653-1659 and Wood, 1985, Biochemical SocietyTransactions 13: 407-410.). CelS2 is a bacterial enzyme that originatesfrom Streptomyces species that are known to grow optimally on cellulosicsubstrates in approximately neutral pH conditions (Kontro et al., 2005,Letters in Applied Microbiology 41: 32-38.). The data presented in FIG.19 show that CelS2 is active at pH 8.0.

Because of the pH-dependent properties of the cellulases, the synergyexperiments reported here were performed at slightly acidic pH, whichmay be suboptimal for the particular CBM33 used (primarily CelS2 fromStreptomyces). It is thus possible that the observed effects of CelS2are smaller than they could be under conditions optimized for CelS2activity.

More generally, an expert in the field will know that natural enzymesvary in terms of their optimum pH and temperatures for activity. Theexpert will know that this will also apply to hydrolytic enzymes, suchas cellulases and chitinases and to oxidohydrolytic enzymes, such asCBP21 and CelS2. It is obvious that for obtaining optimal overallreaction efficiency and/or for maximizing the boosting effect of aCBM33, one needs to take into account the pH optima and temperatureoptima of both the CBM33 and the hydrolytic enzymes. One may obtainenzymes with varying pH and temperature optima by selecting appropriateenzymes from nature or by modification of properties such as pH optimumof natural enzymes using protein engineering type of technologies.

It is conceivable that pH affects the performance of aCBM33+hydrolase(s) type of enzymatic system because pH affects theequilibrium between lactone and the acid form of the oxidized products,which again may affect the efficiency of the hydrolytic enzymes. The pHmay also affect the reductant.

These experiments show that some family 33 CBMs, like CelS2 and E7, areactive on crystalline cellulose, boost cellulase activity in both theabsence and presence of an external electron donor, but showsubstantially higher activity/boosting effect with the electron donorpresent. These observations, as well as the results previously obtainedfor CBP21 acting on chitin, indicate that the oxidation of one of thenewly generated chain ends is important for the function of these CBM33enzymes. That the oxidation indeed also is part of the mechanism forCBM33s that act on cellulose is further supported by data showing thatcyanide, a well known oxygen mimic, inhibits the generation of oxidizedcellooligosaccharides when CelS2 is incubated with AVICEL® in thepresence of an external electron donor (FIG. 22).

Additionally, a variant of CelS2 was generated containing a mutation ofa putatively essential conserved residue, histidine 144, to alanine.This histidine is one of the strongly conserved residues in the metalbinding motif characteristic for family 33 CBMs and corresponds toHis114 in CBP21 (FIGS. 1D and 1F; FIGS. 10A and 10B; Vaaje-Kolstad etal., 2010, Science 330: 219-222). FIGS. 23A and 23B show thatCelS2-H144A was unable to generate oxidized cellooligosaccharides in thepresence of an external electron donor.

It is important to emphasize once more that experiments have beenperformed at only one pH, which is likely to be not optimal for one ofthe two enzyme components (CBM33 or hydrolytic enzyme), as discussedabove. The cellulases used work optimally at the acidic range of the pHscale, while the cellulose oxidating CBM33s are more active at the basicrange of the pH scale. An expert in the field will understand that onemay get different and better results when adapting the pH of thereaction and/or by selecting enzyme variants that are better suited forthe pH used here and to work together at this particular pH. The fullpotential of the oxidative boosting effect may thus not be seen in theseexperiments.

Finally, other cellulosic variants (filter paper and steam explodedsawdust from poplar) were also probed as substrates for CelS2. Solubleoxidized cellooligosaccharides were not observed by MALDI-TOF MSanalysis (results not shown). This is most likely due to the high DP ofthe cellulose in these substrates; cleavages on the crystalline surfaceswill not easily lead to release of soluble (=short)cellooligosaccharides when the overall DP is very high. AVICEL® isspecial in that it is a form of microcrystalline cellulose that has avery low DP (˜60-100; Wallis et al., 1992, Carbohydrate Polymers 17:103-110 and Mormann and U, 2002, Carbohydrate Polymers 50: 349-353)compared to that of the other substrates tested.

However, when the cellulase boosting effect of the CBM33s, in this caseCelS2, was probed with a more “natural” substrate (represented by steamexploded sawdust from poplar), the effect of CelS2 was indeed present asshown by a ˜2-fold increase; FIG. 24). The data shown for the boostedCELLUCLAST™ effect on steam exploded sawdust from poplar, whichrepresents a substrate more like what is used in industrial biomassdegrading applications, also show that CelS2 activity does not dependenton the substrate being of high purity. Clearly, CelS2 also acts oncellulose present in a more natural environment (i.e., embedded in aplant/wood cell wall matrix).

Apart from showing a completely novel enzyme activity for modifyingsolid polysaccharide surfaces, our results point into new directions forenzymatic conversion of recalcitrant polysaccharides. Clearly, CBM33family proteins (such as CBP21, EfCBM33 (FIGS. 15A, 15B, 15C, 15D, and15E), E7 and CelS2 (FIGS. 17-22, 23A, 23B, and 24)) and GH61 familyproteins (such as the GH61 proteins found in T. terrestris,Phanerochaete chrysosporium or Hypocrea jecorina) can dramaticallyincrease the efficiency of hydrolytic enzyme mixtures for chitin andcellulose and a first glimpse of the potential of GH61 proteins forcellulose conversion has indeed been presented very recently (Harris etal., 2010, Biochemistry 49: 3305). The present invention shows how thebeneficial effects of CBM33 and GH61 family proteins may be boosted byadjusting the reaction conditions, i.e., the combined presence of anappropriate metal ion and agents that are reducing and/or generatereactive oxygen species. Clearly, the dependency of theseoxidohydrolases on the presence of molecular oxygen and reductantsacting as external electron donors provides guidelines for processdesign.

Example 2: Effects of CelS2 on Cellulose Degradation by Cellulases, inthe Presence of Reduced Glutathione

FIGS. 20 and 21 show that one of the effects of CelS2 and E7 is toprolong cellulase activity. While reactions with CELLUCLAST™ alone orCel7A alone hardly produce any additional soluble glucose after thefirst time point (24 hours in FIG. 20, 18 hours in FIG. 21), release ofsoluble glucose continues in most cases where a CBM33 protein is presentand this effect is increased in the presence of a reductant. To furtheranalyze this, we conducted a time course experiment to examine theeffect of CelS2 on CELLUCLAST™ activity over time.

Materials and Methods

As in Example 1. Further experimental details, including minordeviations from the standard protocols described in Example 1 areprovided in the figure legends, where necessary, for this Example andthe Examples which follow.

Results

The results (FIG. 25) clearly show that the production of solubleglucose by CELLUCLAST™ levels off, while production of soluble glucosecontinues if CelS2 is present. This effect is increased if a reducingagent is also present. It is conceivable that CelS2 somehow prevents thecellulases from becoming irreversibly and non-productively bound to thesubstrate by modifying the surface of the cellulosic substrate, aphenomenon that is generally considered to reduce cellulase efficiency(Jalak and Väljamäe, 2010, Biotechnology and Bioengineering106:871-883).

Example 3: Effects of CelS2 and Cel7A on Cellulose Degradation byCellulases in the Presence of Reduced Glutathione

To examine the functionality of CelS2, including its effect onprolonging cellulase activity, in more detail, the effect of CelS2 onthe efficiency of a monocomponent cellulase was studied. Themonocomponent enzyme was HjCel7A, obtained by purification from amixture of cellulases from Hypocrea jecorina (Trichoderma reesei).

Materials and Methods

As in Example 1.

Results

The results, depicted in FIG. 26 clearly show that CelS2 boosts theactivity of the cellulase, Cel7A, and that this boosting effect islarger in the presence of a reductant. The results also show that thepresence of CelS2 prolongs the activity of Cel7A.

Example 4: Effects of Different Reductants on CBM33 Efficiency—Studieswith Recombinantly Produced N-Terminal CBM33 Domain of CelS2

To check for the functionality of different reductants the recombinantlyproduced N-terminal CBM33 domain of CelS2 was incubated with 0.8 mM ofreduced glutathione, gallic acid or ascorbic acid and the release ofoxidized oligosaccharides from AVICEL® was monitored,

Materials and Methods

As in Example 1.

Results

FIG. 27 shows that all of the tested reductants boosted the activity ofthe CBM33 protein, although to a different extent. The choice of optimalreductant will, among other things, depend on pH, the substrate and thetype of GH61/CBM33 protein.

Example 5: Effects of Reductant on the Boosting Effect of E7 onCellulase Activity

According to the Pfam bioinformatic analysis (pfam.org), E7 is a singledomain CBM33 protein (Uniprot ID:Q47QG3; E7) whereas CelS2 (Uniprot ID:Q9RJY2) comprises a CBM33 domain with a CBM2 (Carbohydrate-BindingModule 2; see cazy.org) attached on the C-terminal side of the protein.It is important to note that single domain proteins such as E7 areactive by themselves, as illustrated in FIG. 20. To further illustratethis, FIG. 28 was prepared which shows the degradation of cellulose byCELLUCLAST™ or a combination of CELLUCLAST™ and E7 in the presence orabsence of reductant.

Materials and Methods

As in Example 1.

Results

It is clearly seen in FIG. 28 that E7 acts in synergy with CELLUCLAST™,and that the presence of reductants has a boosting effect on E7. Notealso that FIG. 28 shows that one of the effects of E7 is that itprolongs cellulase activity over time, similar to what was observed forCelS2, as described above. FIG. 29 shows a MALDI spectrum of theoxidized products produced by E7 upon incubation with AVICEL® and areductant.

Example 6: Effects of Additional CBMs (the N-Terminal CBM33 Domain ofCelS2 and the C-Terminal CBM2 Domain of CelS2)

As noted in Example 6, E7 is a single domain CBM33 protein, whereasCelS2 comprises a CBM33 domain with a CBM2 attached on the C-terminalside of the protein. It is important to note that single domain proteinssuch as E7 are active by themselves, as illustrated in FIG. 28. Tofurther illustrate this, FIG. 30 was prepared which shows the productsreleased from cellulose by full length CelS2, the recombinantlyexpressed N-terminal CBM33 domain of CelS2, and the recombinantlyexpressed C-terminal CBM2 domain of CelS2.

Materials and Methods

As in Example 1. The sequence of the recombinantly produced N-terminalCBM33 domain is:

(SEQ ID NO: 22) HGVAMMPGSRTYLCQLDAKTGTGALDPTNPACQAALDQSGATALYNWFAVLDSNAGGRGAGYVPDGTLCSAGDRSPYDFSAYNAARSDWPRTHLTSGATIPVEYSNWAAHPGDFRVYLTKPGWSPTSELGWDDLELIQTVTNPPQQGSPGTDGGHYYVVDLALPSGRSGDALIFMQWVRSDSQENFFSCSDVVFDGG.Results

As expected the CBM2 domain alone did not show any activity on cellulose(FIG. 30). However, the CBM33 domain shows activity on AVICEL® withoutits CBM2, although lower than the activity of the whole protein.

These data clearly show that CBM33 domains alone can act synergisticallywith cellulases and that the activity of these single domains isstimulated by the presence of reductants (see also FIG. 27). The dataalso show that the presence of additional CBM domains, such as the CBM2in CelS2, may be beneficial for CBM33 activity. By analogy to what isknown in the scientific literature about the effects of adding varioustypes of CBMs to various types of carbohydrate-active enzymes, it willbe understood that the activity and efficiencies of naturally occurringCBM33 and GH61 proteins may be manipulated by removing, adding orchanging additional CBMs that are fused to the CBM33 or GH61 domain.

Example 7: Metal Activation of CelS2

To identify which is the preferred metal for CelS2, the activity of theN-terminal CBM33 domain of CelS2 was inhibited by EDTA and differentmetals were tested to reactivate the protein.

Materials and Methods

As in Example 1.

Results

FIG. 31 clearly shows that under the conditions used in this experiment,including very low metal concentrations (compared to, e.g., theconcentrations used in FIG. 9) only copper of the tested metals was ableto reactivate CelS2 (10 μM metal concentration in the presence of 20 μMEDTA). Thus, copper ions may be the preferred metal ion for CelS2. Itshould be noted though that at slightly higher (but still low)concentrations, many other metal ions will also work, at least forpractical purposes (Vaaje-Kolstad et al., 2010, supra; Harris et al.,2010, supra; FIG. 9).

Exactly which metal ion is employed by CBM33s and GH61s remains somewhatuncertain, but for practical purposes, several metal ions will work atlow (1 mM range) concentrations. If CBM33s and GH61 prefer copper, thefact that other bivalent metal ions have been observed to activate bothCBM33 and GH61 enzymes (e.g., FIG. 9 and Harris et al., 2010, supra) maybe due to the following: Cu²⁺ ions naturally present in very low amountsin the reaction mixtures (e.g., as part of the substrate) may beinaccessible to the enzyme because they are bound to, e.g., thesubstrate. By adding other bivalent metal ions that tend to bind to thesame “binding sites”, bound copper ions may be released and thus becomeavailable for the enzymes (they are “outcompeted” from the binding sitesby the added bivalent metals). At concentrations in the 1 mM range, thatwork well for other metals, Cu²⁺ inhibits CBM33 action which may be dueto unspecific binding (not shown).

Example 9: Sequence and Activity Comparisons

GH61 enzymes that are known to be active on cellulose (TtGh61E, Harriset al., 2010, supra) or that are likely to be active on cellulose byanalogy, such as the two GH61 proteins encoded on the genome of Hypocreajecorina (or Trichoderma reesei; HjGH61A & HjGH61B) share severalconserved residues in addition to the two histidines making up the metalbinding site (FIGS. 32A and 32B). Interestingly, these same residues areconserved in cellulose-active E7, whereas several of them are absent inthe chitin-active CBP21 (FIGS. 33A, 33B, and 33C).

Example 10: Cleavage of Cellulose by GH61 Proteins in the Presence ofReductants

GH61 proteins TtGH61E (SEQ ID NO: 1) and TaGH61A (SEQ ID NO: 2) wereincubated with cellulose and ascorbic acid to demonstrate that theseproteins can cleave cellulose and yield oxidized products in thepresence of ascorbic acid. The two proteins were cloned and produced asdescribed in Harris et al., 2010, supra. FIG. 34 clearly shows thatTtGH61E cuts cellulose producing oxidized and nativecelluoligosaccharides in a similar way as CBM33 proteins. While theproduct pattern is similar to that obtained with CBM33s, it is notidentical. The patterns typically obtained with CBM33s acting oncellulose in the presence of reductants (FIGS. 19, 22, 27, 30, and 31)show different peridiodicities (i.e., relative abundances of the variousproducts, both oxidized and native oligosaccharides). This may indicatethat TtGH61E acts on the substrate in a slightly different way, e.g., byattacking another face of the crystal. Clearly, however, the overalloutcome of the reaction of TtGH61E in the presence of the reductant issimilar to the outcome of reactions of CBM33s on cellulose in thepresence of reductant. FIG. 34 also shows that TaGH61A is much lessactive than TtGH61E, at least on this substrate, the production ofoxidized sugars being very low. Notably, this enzyme produces relativelymore native cello-oligosaccharides, i.e., a product pattern that isclearly different from the product patterns obtained with TtGH61E andthe CBM33s, which again may indicate slight variations in substratebinding specificity.

Example 11: Effect of Reductants on Cellic™ CTec2, a CellulasePreparation Containing GH61

Since the commercially available cellulase preparation Cellic™ CTec2contains GH61 proteins, the effect of ascorbic acid on the efficiency ofthis enzyme preparation was tested. In this experiment the effect ofascorbic acid on glucose release from cellulose by Cellic™ CTec2 wasinvestigated. FIG. 35 shows that the presence of ascorbic acid increasedthe release of glucose from both Filter Paper and AVICEL® by about 30%,demonstrating the beneficial effects of ascorbic acid.

What is claimed is:
 1. A process for producing a fermentation product,comprising: (a) saccharifying a cellulosic material with an enzymecomposition comprising a GH61 protein and one or more enzymes selectedfrom the group consisting of an endoglucanase, a cellobiohydrolase, abeta-glucosidase, and a CBM33, wherein the saccharifying is carried outin the presence of at least one reducing agent and at least one divalentmetal ion, wherein the at least one reducing agent is selected from thegroup consisting of ascorbic acid, coumaric acid, ferulic acid, gallicacid, glucose, glucosamine, N-acetylglucosamine, reduced glutathione,humic acid, succinic acid, and lignin or fragments thereof, wherein theat least one divalent metal ion is selected from the group consisting ofCa²⁺, Co²⁺, Cu²⁺, Mg²⁺, Mn²⁺, Ni²⁺, and Zn²⁺, and wherein thesaccharifying of the cellulosic material is increased by the presencethe GH61 protein in combination with the at least one reducing agent andthe at least one divalent metal ion relative to without the combination;(b) fermenting the saccharified cellulosic material with one or morefermenting microorganisms to produce the fermentation product; and (c)recovering the fermentation product from the fermentation.
 2. Theprocess of claim 1, wherein the cellulosic material is pretreated. 3.The process of claim 1, wherein the enzyme composition further comprisesone or more enzymes selected from the group consisting of an esterase,an expansin, a hemicellulase, a laccase, a ligninolytic enzyme, apectinase, a peroxidase, a protease, and a swollenin.
 4. The process asclaimed in claim 1, wherein the hemicellulase is one or more enzymesselected from the group consisting of an acetylxylan esterase, anarabinofuranosidase, a feruloyl esterase, a glucuronidase, a mannanase,a xylanase, and a xylosidase.
 5. The process of claim 1, wherein steps(a) and (b) are performed simultaneously in a simultaneoussaccharification and fermentation.
 6. The process of claim 1, whereinthe fermentation product is an alcohol, an alkane, a cycloalkane, analkene, an amino acid, a gas, isoprene, a ketone, an organic acid, orpolyketide.
 7. The process of claim 1, wherein said reducing agent isascorbic acid.
 8. The process of claim 1, wherein said divalent metalion is Cu²⁺.
 9. The method of claim 1, wherein the GH61 proteincomprises the amino acid sequence of SEQ ID NO: 1, 2, 3, 15, or 16, or asequence with at least 90% sequence identity thereto.
 10. The method ofclaim 1, wherein the GH61 protein comprises the amino acid sequence ofSEQ ID NO: 1, 2, 3, 15, or 16.